Camptothecine antibody-drug conjugates and methods of use thereof

ABSTRACT

The present disclosure provides antibody-drug conjugate (ADC) structures, which include a camptothecine or a camptothecine derivative linked to a polypeptide (e.g., an antibody) through a linker. The disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/237,355, filed Aug. 26, 2021, U.S. Provisional Application No.63/214,525, filed Jun. 24, 2021, U.S. Provisional Application No.63/186,489, filed May 10, 2021, and U.S. Provisional Application No.63/138,182, filed Jan. 15, 2021, the disclosures of which areincorporated herein by reference.

INTRODUCTION

The field of protein-small molecule therapeutic conjugates has advancedgreatly, providing a number of clinically beneficial drugs with thepromise of providing more in the years to come. Protein-conjugatetherapeutics can provide several advantages, due to, for example,specificity, multiplicity of functions and relatively low off-targetactivity, resulting in fewer side effects.

SUMMARY

Camptothecines are a family of antitumor agents sharing commonstructural core i (FIG. 1, panel A). Due to their ability to inhibit theactivity of DNA Topoisomerase I, an intracellular enzyme essential forcell replication, several synthetic and semi-synthetic camptothecineshave been used as small-molecule cancer chemotherapies (e.g. topotecanand irinotecan). In contrast, more potent analogs, such as SN-38 (1) andExatecan (3) (FIG. 1, panel B), show significant off-target toxicitiesthat prevent using them directly to treat cancers. Targeted delivery ofhighly potent camptothecines to tumor tissue may circumvent the toxicityproblem and offer a more well-tolerated therapy. The present disclosuredescribes the preparation of antibody-drug conjugates (ADCs) containingcamptothecines of general structure i (FIG. 1, panel A). In eachconjugate, the cytotoxin of choice is connected to the antibody (e.g.,mAb) through a cleavable linker attached to either the common C20alcohol, or through various chemical handles specific to particularcamptothecine analogs (FIG. 1).

The present disclosure provides antibody-drug conjugate (ADC)structures, which include a camptothecine or a camptothecine derivativelinked to a polypeptide (e.g., an antibody) through a linker. Thedisclosure also encompasses compounds and methods for production of suchconjugates, as well as methods of using the conjugates.

Aspects of the present disclosure include a conjugate of formula (I):

wherein:

Z is CR¹⁰ or N,

R⁷ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R⁸ and R⁹ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁸ and R⁹ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

each R¹⁰ is independently selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

W is a polypeptide;

L is a linker attached to a compound of formula (II) at R¹, R², R³, R⁴,R⁵ or R⁶:

R¹ and R² are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R¹ and R² are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R³ and R⁴ are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R³ and R⁴ are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R⁵ is selected from hydrogen, halogen, hydroxy, amino, substitutedamino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

R⁶ is selected from OH and OC(O)R¹¹; and

R¹¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl,

wherein at least one R¹⁰ is optionally linked to a second compound offormula (II).

In some embodiments, the compound of formula (II) has the structure offormula (IIa):

wherein R³ is OH and L is attached at R⁶; or L is attached at R³ and R⁶is OH; or wherein the compound of formula (II) has the structure offormula (IIb):

wherein R^(1a) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1a) and R⁶ is OH; or

wherein the compound of formula (II) has the structure of formula (IIc):

wherein R^(1b) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1b) and R⁶ is OH; or

wherein the compound of formula (II) has the structure of formula (IId):

wherein R^(2a) and R^(2b) are each independently selected from H, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl,and L is attached at R⁶; or L is attached at R^(2a) or R^(2b) and R⁶ isOH; or

wherein the compound of formula (II) has the structure of formula (IIe):

wherein R^(2c) is selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxylester, acyl, and sulfonyl, and attachment to L is indicated by the wavyline.

In some embodiments, L comprises:

-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)-(T⁶-V⁶)_(f)-,

wherein

a, b, c, d, e and f are each independently 0 or 1;

T¹, T², T³, T⁴, T⁵ and T⁶ are each independently selected from acovalent bond, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl,(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine(4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC),para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC),para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl(PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, adisulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEGis a polyethylene glycol, and AA is an amino acid residue or an aminoacid analog, wherein each w is an integer from 1 to 20, each n is aninteger from 1 to 30, each p is an integer from 1 to 20, and each m isan integer from 1 to 12;

V¹, V², V³, V⁴, V⁵ and V⁶ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and —P(O)OH—, wherein each q is an integerfrom 1 to 6;

each R¹³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, and substituted aryl; and

each R¹⁵ is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In some embodiments, L is a linker wherein:

T¹ is selected from a (C₁-C₁₂)alkyl and a substituted (C₁-C₁₂)alkyl;

T², T³, T⁴, T⁵ and T⁶ are each independently selected from a covalentbond, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine (4AP), MABO, MABC, PABO,PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester;and

V¹, V², V³, V⁴, V⁵ and V⁶ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂—, and —P(O)OH—;

wherein:

(PEG)_(n) is

where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4AP) is

and

each R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a polyethylene glycol moiety, an aryl and asubstituted aryl, wherein any two adjacent R¹² groups may be cyclicallylinked to form a piperazinyl ring.

In some embodiments, T¹, T², T³, T⁴, T⁵ and T⁶ are each optionallysubstituted with a glycoside. In some embodiments, MABO, MABC, PABO,PABC, PAB, PABA, PAP and PHP are each optionally substituted with aglycoside. In some embodiments, the glycoside is selected from aglucuronide, a galactoside, a glucoside, a mannoside, a fucoside,O-GlcNAc, and O-GalNAc.

In some embodiments, L is a linker wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)n and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

T⁶ is EDA and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is absent and V⁵ is —NR¹⁵(C₆H₄)—; and

T⁶ is absent and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is —NR¹⁵—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent;

T⁴ is EDA and V⁴ is —CO—; and

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent; and

d, e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABA and V⁵ is —CO—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —SO₂—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is substituted (C₁-C₁₂)alkyl and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

T⁵ is (C₁-C₁₂)alkyl and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —O—;

T⁴ is (C₁-C₁₂)alkyl and V⁴ is —CO—;

T⁵ is AA and V⁵ is absent;

T⁶ is PABC and V⁶ is absent; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is absent;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CONH—;

T³ is substituted (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABO and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PAP and V⁵ is —COO—; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PAP and V⁴ is —COO—; and

e and f are each 0.

In some embodiments, one R¹⁰ is linked via a second linker, L^(B), to asecond compound of formula (II).

In some embodiments, L^(B) comprises:

-(T⁷-V⁷)_(g)-(T⁸-V⁸)_(h)-(T⁹-V⁹)_(i)-(T¹⁰-V¹⁰)_(j)-(T¹¹-V¹¹)_(k)-(T¹²-V¹²)_(l)-,

wherein

g, h, i, j, k and l are each independently 0 or 1;

T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² are each independently selected from acovalent bond, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl,(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine(4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC),para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC),para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl(PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, adisulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEGis a polyethylene glycol, and AA is an amino acid residue or an aminoacid analog, wherein each w is an integer from 1 to 20, each n is aninteger from 1 to 30, each p is an integer from 1 to 20, and each m isan integer from 1 to 12;

V⁷, V⁸, V⁹, V¹⁰, V¹¹ and V¹² are each independently selected from thegroup consisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and —P(O)OH—, wherein each q is an integerfrom 1 to 6;

each R¹³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, and substituted aryl; and

each R¹⁵ is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In some embodiments, wherein T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² are eachoptionally substituted with a glycoside. In some embodiments, MABO,MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substitutedwith a glycoside. In some embodiments, the glycoside is selected from aglucuronide, a galactoside, a glucoside, a mannoside, a fucoside,O-GlcNAc, and O-GalNAc.

In some embodiments, L^(B) is a linker wherein:

T⁷ is absent and V⁷ is —NR¹⁵CO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is EDA and V¹¹ is —CO—; and

l is 0; or

wherein:

T⁷ is absent and V⁷ is —NR¹⁵CO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent; and

k and l are each 0; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is an amino acid analog and V⁹ is —NH—;

T¹⁰ is (PEG)_(n) and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent; and

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CONH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent; and

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is substituted (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is (C₁-C₁₂)alkyl and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —O—;

T¹⁰ is (C₁-C₁₂)alkyl and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent;

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is an amino acid analog and V⁸ is absent;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CONH—;

T⁹ is substituted (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is AA and V⁸ is —NH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PAP and V¹⁰ is —COO—; and

k and l are each 0.

In some embodiments, the conjugate is selected from:

Aspects of the present disclosure include a compound of formula (III):

wherein:

Z is CR¹⁰ or N,

R⁸ and R⁹ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁸ and R⁹ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

each R¹⁰ is independently selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is a linker attached to a compound of formula (II) at R¹, R², R³, R⁴,R⁵ or R⁶:

R¹ and R² are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R¹ and R² are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R³ and R⁴ are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R³ and R⁴ are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R⁵ is selected from hydrogen, halogen, hydroxy, amino, substitutedamino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

R⁶ is selected from OH and OC(O)R¹¹; and

R¹¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl,

wherein at least one R¹⁰ is optionally linked to a second compound offormula (II).

In some embodiments, the compound of formula (II) has the structure offormula (IIa):

wherein R³ is OH and L is attached at R⁶; or L is attached at R³ and R⁶is OH; or wherein the compound of formula (II) has the structure offormula (IIb):

wherein R^(1a) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1a) and R⁶ is OH; or

wherein the compound of formula (II) has the structure of formula (IIc):

wherein R^(1b) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1b) and R⁶ is OH; or

wherein the compound of formula (II) has the structure of formula (IId):

wherein R^(2a) and R^(2b) are each independently selected from H, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl,and L is attached at R⁶; or L is attached at R^(2a) or R^(2b) and R⁶ isOH; or

wherein the compound of formula (II) has the structure of formula (IIe):

wherein R^(2c) is selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxylester, acyl, and sulfonyl, and attachment to L is indicated by the wavyline.

In some embodiments, L comprises:

-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)-(T⁶-V⁶)_(f)-,

wherein

a, b, c, d, e and f are each independently 0 or 1;

T¹, T², T³, T⁴, T⁵ and T⁶ are each independently selected from acovalent bond, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl,(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine(4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC),para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC),para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl(PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, adisulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEGis a polyethylene glycol, and AA is an amino acid residue or an aminoacid analog, wherein each w is an integer from 1 to 20, each n is aninteger from 1 to 30, each p is an integer from 1 to 20, and each m isan integer from 1 to 12;

V¹, V², V³, V⁴, V⁵ and V⁶ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and —P(O)OH—, wherein each q is an integerfrom 1 to 6;

each R¹³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, and substituted aryl; and

each R¹⁵ is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In some embodiments, L is a linker wherein:

T¹ is selected from a (C₁-C₁₂)alkyl and a substituted (C₁-C₁₂)alkyl;

T², T³, T⁴, T⁵ and T⁶ are each independently selected from a covalentbond, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine (4AP), MABO, MABC, PABO,PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester;and

V¹, V², V³, V⁴, V⁵ and V⁶ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂—, and —P(O)OH—;

wherein:

(PEG)_(n) is

where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4AP) is

and

each R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a polyethylene glycol moiety, an aryl and asubstituted aryl, wherein any two adjacent R¹² groups may be cyclicallylinked to form a piperazinyl ring.

In some embodiments, T¹, T², T³, T⁴, T⁵ and T⁶ are each optionallysubstituted with a glycoside. In some embodiments, MABO, MABC, PABO,PABC, PAB, PABA, PAP and PHP are each optionally substituted with aglycoside. In some embodiments, the glycoside is selected from aglucuronide, a galactoside, a glucoside, a mannoside, a fucoside,O-GlcNAc, and O-GalNAc.

In some embodiments, L is a linker wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

T⁶ is EDA and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is absent and V⁵ is —NR¹⁵(C₆H₄)—; and

T⁶ is absent and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is —NR¹⁵—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent;

T⁴ is EDA and V⁴ is —CO—; and

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent; and

d, e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABA and V⁵ is —CO—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —SO₂—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABA and V⁵ is —CO—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —SO₂—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is substituted (C₁-C₁₂)alkyl and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

T⁵ is (C₁-C₁₂)alkyl and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —O—;

T⁴ is (C₁-C₁₂)alkyl and V⁴ is —CO—;

T⁵ is AA and V⁵ is absent;

T⁶ is PABC and V⁶ is absent; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is absent;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CONH—;

T³ is substituted (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABO and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PAP and V⁵ is —COO—; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PAP and V⁴ is —COO—; and

e and f are each 0.

In some embodiments, one R¹⁰ is linked via a second linker, L^(B), to asecond compound of formula (II).

In some embodiments, L^(B) comprises:

-(T⁷-V⁷)_(g)-(T⁸-V⁸)_(h)-(T⁹-V⁹)_(i)-(T¹⁰-V¹⁰)_(j)-(T¹¹-V¹¹)_(k)-(T¹²-V¹²)_(l)-,

wherein

g, h, i, j, k and l are each independently 0 or 1;

T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² are each independently selected from acovalent bond, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl,(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine(4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC),para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC),para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl(PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, adisulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEGis a polyethylene glycol, and AA is an amino acid residue or an aminoacid analog, wherein each w is an integer from 1 to 20, each n is aninteger from 1 to 30, each p is an integer from 1 to 20, and each m isan integer from 1 to 12;

V⁷, V⁸, V⁹, V¹⁰, V¹¹ and V¹² are each independently selected from thegroup consisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and —P(O)OH—, wherein each q is an integerfrom 1 to 6;

each R¹³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, and substituted aryl; and

each R¹⁵ is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In some embodiments, T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² are each optionallysubstituted with a glycoside. In some embodiments, MABO, MABC, PABO,PABC, PAB, PABA, PAP and PHP are each optionally substituted with aglycoside. In some embodiments, the glycoside is selected from aglucuronide, a galactoside, a glucoside, a mannoside, a fucoside,O-GlcNAc, and O-GalNAc.

In some embodiments, L^(B) is a linker wherein:

T⁷ is absent and V⁷ is —NR¹⁵CO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is EDA and V¹¹ is —CO—; and

l is 0; or

wherein:

T⁷ is absent and V⁷ is —NR¹⁵CO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent; and

k and l are each 0; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is an amino acid analog and V⁹ is —NH—;

T¹⁰ is (PEG)_(n) and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent; and

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CONH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent; and

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is substituted (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is (C₁-C₁₂)alkyl and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —O—;

T¹⁰ is (C₁-C₁₂)alkyl and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent;

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is an amino acid analog and V⁸ is absent;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CONH—;

T⁹ is substituted (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is AA and V⁸ is —NH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PAP and V¹⁰ is —COO—; and

k and l are each 0.

In some embodiments, the compound is selected from:

Aspects of the present disclosure include a pharmaceutical compositioncomprising a conjugate according to the present disclosure, and apharmaceutically-acceptable excipient.

Aspects of the present disclosure include a method comprisingadministering to a subject an effective amount of a conjugate accordingto the present disclosure.

Aspects of the present disclosure include a method of treating cancer ina subject by administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising a conjugate accordingto the present disclosure, where the administering is effective to treatcancer in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows chemical structures of the camptothecine family oftopoisomerase I inhibitors. (FIG. 1, panel A) General structure ofcamptothecines. (FIG. 1, panel B) Representative examples ofcamptothecines to be used as payloads for antibody-drug conjugates(arrows indicate modification sites for linker attachment).

FIG. 2 shows a schematic of the HIPS ligation for the synthesis of ADCs.Antibodies carrying aldehyde moieties are reacted with aHydrazino-iso-Pictet-Spengler (HIPS) linker and payload to generate asite-specifically conjugated ADC with a stable azacarboline linkage.

FIG. 3 shows a polymeric reversed phase column (PLRP) trace of compound12, conjugate 2941, which included two tag sites and yielded a DAR of2.75, 2.2% HMW.

FIG. 4 shows an analytical size exclusion chromatography (SEC) trace ofcompound 12, conjugate 2941, which included two tag sites and yielded aDAR of 2.75, 2.2% HMW.

FIG. 5 shows a PLRP trace of compound 12, conjugate 2944, which includedtwo tag sites and yielded a DAR of 3.12, 4.5% HMW.

FIG. 6 shows an SEC trace of compound 12, conjugate 2944, which includedtwo tag sites and yielded a DAR of 3.12, 4.5% HMW.

FIG. 7 shows a PLRP trace of compound 12, conjugate 2746, which includedtwo tag sites and yielded a DAR of 2.78, 3.4% HMW.

FIG. 8 shows a hydrophobic interaction column (HIC) trace of compound12, conjugate 2746, which included two tag sites and yielded a DAR of2.78, 3.4% HMW.

FIG. 9 shows an SEC trace of compound 12, conjugate 2746, which includedtwo tag sites and yielded a DAR of 2.78, 3.4% HMW.

FIG. 10 shows a PLRP trace of compound 12, conjugate 2749, whichincluded two tag sites and yielded a DAR of 3.04, 0.9% HMW.

FIG. 11 shows an SEC trace of compound 12, conjugate 2749, whichincluded two tag sites and yielded a DAR of 3.04, 0.9% HMW.

FIG. 12 shows a PLRP trace of compound 12, conjugate 2752, whichincluded two tag sites and yielded a DAR of 3.01, 4.3% HMW.

FIG. 13 shows an HIC trace of compound 12, conjugate 2752, whichincluded two tag sites and yielded a DAR of 3.01, 4.3% HMW.

FIG. 14 shows an SEC trace of compound 12, conjugate 2752, whichincluded two tag sites and yielded a DAR of 3.01, 4.3% HMW.

FIG. 15 shows a PLRP trace of compound 12, conjugate 2755, whichincluded two tag sites and yielded a DAR of 2.72, 3.2% HMW.

FIG. 16 shows an HIC trace of compound 12, conjugate 2755, whichincluded two tag sites and yielded a DAR of 2.72, 3.2% HMW.

FIG. 17 shows an SEC trace of compound 12, conjugate 2755, whichincluded two tag sites and yielded a DAR of 2.72, 3.2% HMW.

FIG. 18 shows an HIC trace of compound 12, conjugate 2758, whichincluded one tag site and yielded a DAR of 1.54, 1.6% HMW.

FIG. 19 shows an SEC trace of compound 12, conjugate 2758, whichincluded one tag site and yielded a DAR of 1.54, 1.6% HMW.

FIG. 20 shows a PLRP trace of compound 12, conjugate 2762, whichincluded two tag sites and yielded a DAR of 2.95, 3.6% HMW.

FIG. 21 shows an SEC trace of compound 20, conjugate 2762, whichincluded two tag sites and yielded a DAR of 2.95, 3.6% HMW.

FIG. 22 shows a PLRP trace of compound 20, conjugate 2765, whichincluded two tag sites and yielded a DAR of 2.97, 1.2% HMW.

FIG. 23 shows an SEC trace of compound 20, conjugate 2765, whichincluded two tag sites and yielded a DAR of 2.97, 1.2% HMW.

FIG. 24 shows a PLRP trace of compound 20, conjugate 2768, whichincluded two tag sites and yielded a DAR of 2.92, 5.3% HMW.

FIG. 25 shows an SEC trace of compound 20, conjugate 2768 which includedtwo tag sites and yielded a DAR of 2.92, 5.3% HMW.

FIG. 26 shows a PLRP trace of compound 20, conjugate 2771 which includedtwo tag sites and yielded a DAR of 2.89, 4.3% HMW.

FIG. 27 shows an SEC trace of compound 20, conjugate 2771 which includedtwo tag sites and yielded a DAR of 2.89, 4.3% HMW.

FIG. 28 shows an HIC trace of compound 20, conjugate 2774, whichincluded one tag site and yielded a DAR of 1.22, 1.8% HMW.

FIG. 29 shows an SEC trace of compound 20, conjugate 2774, whichincluded one tag site and yielded a DAR of 1.22, 1.8% HMW.

FIG. 30 shows a PLRP trace of compound 27, conjugate 2763, whichincluded two tag sites and yielded a DAR of 2.43, 6.0% HMW.

FIG. 31 shows an SEC trace of compound 27, conjugate 2763, whichincluded two tag sites and yielded a DAR of 2.43, 6.0% HMW.

FIG. 32 shows a PLRP trace of compound 27, conjugate 2766, whichincluded two tag sites and yielded a DAR of 2.43, 2.2% HMW.

FIG. 33 shows an SEC trace of compound 27, conjugate 2766, whichincluded two tag sites and yielded a DAR of 2.43, 2.2% HMW.

FIG. 34 shows a PLRP trace of compound 27, conjugate 2769, whichincluded two tag sites and yielded a DAR of 2.42, 8.2% HMW.

FIG. 35 shows an SEC trace of compound 27, conjugate 2769, whichincluded two tag sites and yielded a DAR of 2.42, 8.2% HMW.

FIG. 36 shows a PLRP trace of compound 27, conjugate 2772, whichincluded two tag sites and yielded a DAR of 2.47, 6.0% HMW.

FIG. 37 shows an SEC trace of compound 27, conjugate 2772, whichincluded two tag sites and yielded a DAR of 2.47, 6.0% HMW.

FIG. 38 shows an HIC trace of compound 27, conjugate 2775, whichincluded one tag site and yielded a DAR of 0.45, 1.4% HMW.

FIG. 39 shows an SEC trace of compound 27, conjugate 2775, whichincluded one tag site and yielded a DAR of 0.45, 1.4% HMW.

FIG. 40 shows a PLRP trace of compound 34, conjugate 2942, whichincluded two tag sites and yielded a DAR of 2.78, 11.2% HMW.

FIG. 41 shows an SEC trace of compound 34, conjugate 2942, whichincluded two tag sites and yielded a DAR of 2.78, 11.2% HMW.

FIG. 42 shows a PLRP trace of compound 34, conjugate 2945, whichincluded two tag sites and yielded a DAR of 3.03, 5.2% HMW.

FIG. 43 shows an SEC trace of compound 34, conjugate 2945, whichincluded two tag sites and yielded a DAR of 3.03, 5.2% HMW.

FIG. 44 shows a PLRP trace of compound 42, conjugate 2943, whichincluded two tag sites and yielded a DAR of 5.44, 21.3% HMW.

FIG. 45 shows an SEC trace of compound 42, conjugate 2943, whichincluded two tag sites and yielded a DAR of 5.44, 21.3% HMW.

FIG. 46 shows a PLRP trace of compound 42, conjugate 2946, whichincluded two tag sites and yielded a DAR of 5.07, 15.3% HMW.

FIG. 47 shows an SEC trace of compound 42, conjugate 2946, whichincluded two tag sites and yielded a DAR of 5.07, 15.3% HMW.

FIG. 48 shows a PLRP trace of compound 45, conjugate 2748, whichincluded two tag sites and yielded a DAR of 3.51, 3.6% HMW.

FIG. 49 shows an HIC trace of compound 45, conjugate 2748, whichincluded two tag sites and yielded a DAR of 3.51, 3.6% HMW.

FIG. 50 shows an SEC trace of compound 45, conjugate 2748, whichincluded two tag sites and yielded a DAR of 3.51, 3.6% HMW.

FIG. 51 shows a PLRP trace of compound 45, conjugate 2751, whichincluded two tag sites and yielded a DAR of 2.98, 2.1% HMW.

FIG. 52 shows an SEC trace of compound 45, conjugate 2751, whichincluded two tag sites and yielded a DAR of 2.98, 2.1% HMW.

FIG. 53 shows a PLRP trace of compound 45, conjugate 2754, whichincluded two tag sites and yielded a DAR of 3.5, 2.3% HMW.

FIG. 54 shows an HIC trace of compound 45, conjugate 2754, whichincluded two tag sites and yielded a DAR of 3.5, 2.3% HMW.

FIG. 55 shows an SEC trace of compound 45, conjugate 2754, whichincluded two tag sites and yielded a DAR of 3.5, 2.3% HMW.

FIG. 56 shows a PLRP trace of compound 45, conjugate 2757, whichincluded two tag sites and yielded a DAR of 3.69, 2.0% HMW.

FIG. 57 shows an SEC trace of compound 45, conjugate 2757, whichincluded two tag sites and yielded a DAR of 3.69, 2.0% HMW.

FIG. 58 shows an HIC trace of compound 45, conjugate 2760, whichincluded one tag site and yielded a DAR of 1.65, 0.8% HMW.

FIG. 59 shows an SEC trace of compound 45, conjugate 2760, whichincluded one tag site and yielded a DAR of 1.65, 0.8% HMW.

FIG. 60 shows a PLRP trace of compound 47, conjugate 3065, whichincluded two tag sites and yielded a DAR of 6.82, 1.8% HMW.

FIG. 61 shows an SEC trace of compound 47, conjugate 3065, whichincluded two tag sites and yielded a DAR of 6.82, 1.8% HMW.

FIG. 62 shows a PLRP trace of compound 47, conjugate 3066, whichincluded two tag sites and yielded a DAR of 4.48.

FIG. 63 shows an HIC trace of compound 47, conjugate 3067, whichincluded one tag site and yielded a DAR of 3.63, 4.4% HMW.

FIG. 64 shows an SEC trace of compound 47, conjugate 3067, whichincluded one tag site and yielded a DAR of 3.63, 4.4% HMW.

FIG. 65 shows an HIC trace of compound 47, conjugate 3068, whichincluded one tag site and yielded a DAR of 3.52, 0.8% HMW.

FIG. 66 shows an SEC trace of compound 47, conjugate 3068, whichincluded one tag site and yielded a DAR of 3.52, 0.8% HMW.

FIG. 67 shows a PLRP trace of compound 56, conjugate 3063, whichincluded two tag sites and yielded a DAR of 3.35, 3.3% HMW.

FIG. 68 shows an SEC trace of compound 56, conjugate 3063, whichincluded two tag sites and yielded a DAR of 3.35, 3.3% HMW.

FIG. 69 shows a PLRP trace of compound 56, conjugate 3064, whichincluded two tag sites and yielded a DAR of 2.8.

FIG. 70 shows a graph of in vitro cytotoxicity assays of freetopoisomerase inhibitors in NCI-N87 gastric cancer cells.

FIG. 71 shows a graph of in vitro cytotoxicity assays of freetopoisomerase inhibitors in Sk-Br-3 breast cancer cells.

FIG. 72 shows a graph of in vitro cytotoxicity assays of freetopoisomerase inhibitors in Granta NHL cells.

FIG. 73 shows a graph of in vitro cytotoxicity assays of freetopoisomerase inhibitors in MDA-MB-468 breast cancer cells.

FIG. 74 shows a graph of in vitro cytotoxicity assays of freetopoisomerase inhibitors in MDA-PCa-2b prostate cancer cells.

FIG. 75 shows a graph of in vitro cytotoxicity assays in MDA-MB-468breast cancer cells of a TROP-2 targeted ADC made using Compound 61,according to embodiments of the present disclosure.

FIG. 76 shows a graph of in vitro cytotoxicity assays in NCI-N87 gastriccancer cells of a HER2 targeted ADC made using Compound 61, according toembodiments of the present disclosure.

FIG. 77 shows a graph of in vitro cytotoxicity assays in SU-DHL-1 ALCLcells of a CD25 targeted ADC made using Compound 61, according toembodiments of the present disclosure.

FIG. 78 shows a graph of in vitro cytotoxicity assays in BxPC-3pancreatic cancer cells of a TROP-2 targeted ADC made using Compound 61,according to embodiments of the present disclosure.

FIG. 79 shows a graph of in vitro cytotoxicity assays in NCI-N87 gastriccancer cells of a HER2 targeted ADC made using Compound 65, according toembodiments of the present disclosure.

FIG. 80 shows a graph of in vitro cytotoxicity assays in Sk-Br-3 breastcancer cells of a HER2 targeted ADC made using Compound 65, according toembodiments of the present disclosure.

FIG. 81 shows a schematic drawing of ELISA assays used to determinetotal antibody and ADC concentrations for pharmacokinetic (PK) sampleanalysis.

FIG. 82 shows a graph of concentration (μg/mL) vs. days post dosefollowing a 0.9 mg/kg dose of trastuzumab antibody.

FIG. 83 shows a graph of concentration (μg/mL) vs. days post dosefollowing a 0.9 mg/kg dose of a conventional HER2 topoisomeraseinhibitor conjugated ADC bearing a protease cleavable linker.

FIG. 84 shows a graph of concentration (μg/mL) vs. days post dosefollowing a 0.9 mg/kg dose of CH1-3/CT-tagged trastuzumab conjugated toconstruct 61, according to embodiments of the present disclosure.

FIG. 85 shows a graph of concentration (μg/mL) vs. days post dosefollowing a 0.9 mg/kg dose of CH1-3/CT-tagged trastuzumab conjugated toconstruct 65, according to embodiments of the present disclosure.

FIG. 86 shows a graph of mean tumor volume (mm³) vs. days, whichindicates in vivo efficacy against an NCI-H292 xenograft of TROP-2targeted ADCs carrying topoisomerase inhibitor payloads. n=8 mice/group;dosing is indicated by arrows.

FIG. 87 shows a graph of mean tumor volume (mm³) vs. days, whichindicates in vivo efficacy against an NCI-H292 xenograft of TROP-2targeted ADCs carrying topoisomerase inhibitor payloads. n=7 mice/group.A single i.v. dose was delivered on Day 0.

FIG. 88 shows a graph of mean tumor volume (mm³) vs. days, whichindicates in vivo efficacy against an NCI-H1781 xenograft of nectin-4targeted ADCs carrying topoisomerase inhibitor payloads. n=5 mice/group.A 5 mg/kg dose was delivered i.v. on Days 0 and 7.

FIG. 89 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (165), (65), (175) as compared to (1) or (2)against MBA-MB-468 cells.

FIG. 90 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (165), (65), (175) as compared to (1) or (2)against BxPC-3 cells.

FIG. 91 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (65), (67), or (73) as compared to (2) againstSK-BR-3 cells.

FIG. 92 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (65), (67), or (73) as compared to (2) againstNCI-N87 cells.

FIG. 93 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (65) or (80) as compared to (2) against MDA-MB-468cells.

FIG. 94 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (65) or (80) as compared to (2) against BxPC-3cells.

FIG. 95 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (99), (103), or (110) as compared to (2) againstNCI-N87 cells.

FIG. 96 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (65), (86), or (92) as compared to (2) againstSK-BR-3 cells.

FIG. 97 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (65), Trodelvy, or a CL2A-SN38 isotype controlconjugate as compared to (1) or (2) against SK-BR-3 cells.

FIG. 98 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (65), Enhertu, or a MC-GGFG-Dxd isotype controlconjugate as compared to (2) against NCI-N87 cells.

FIG. 99 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (65) or (113) as compared to (2) againstMDA-MB-468 cells.

FIG. 100 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (65), (136), or (142) as compared to (2) againstNCI-N87 cells.

FIG. 101 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (65), (127), or (131) as compared to (2) againstMDA-MB-468 cells.

FIG. 102 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (65), (127), or (131) as compared to (2) againstNCI-N87 cells.

FIG. 103 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (151), (147), or (131) as compared to (2), (148),or (144) against NCI-N87 cells.

FIG. 104 shows a graph of in vitro potency of HER2 targeted or isotypecontrol ADCs carrying (151), (147), or (131) as compared to (2), (148),or (144) against SK-BR-3 cells.

FIG. 105 shows a graph of in vitro potency of TROP-2 targeted or isotypecontrol ADCs carrying (155) or CL2A-SN38 as compared to (1) againstMDA-MB-468 cells.

FIG. 106 shows a graph of Compound 127 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 7.15 as determined by PLRP.

FIG. 107 shows a graph of Compound 131 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 6.80 as determined by PLRP.

FIG. 108 shows a graph of Compound 127 CH1-3/CT-tagged trastuzumabconjugate is 94.4% monomeric as determined by analytical SEC.

FIG. 109 shows a graph of Compound 131 CH1-3/CT-tagged trastuzumabconjugate is 93.6% monomeric as determined by analytical SEC.

FIG. 110 shows a graph of Compound 165 CH1-3/CT-tagged sacituzumabconjugate yields a DAR of 3.41 as determined by HIC.

FIG. 111 shows a graph of Compound 165 CH1-3/CT-tagged sacituzumabconjugate is 98.2% monomeric as determined by analytical SEC.

FIG. 112 shows a graph of Compound 165 CH1-3/CT-tagged polatuzumabconjugate yields a DAR of 3.67 as determined by HIC.

FIG. 113 shows a graph of Compound 165 CH1-3/CT-tagged polatuzumabconjugate is 97.4% monomeric as determined by analytical SEC.

FIG. 114 shows a graph of Compound 80 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 5.86 as determined by PLRP.

FIG. 115 shows a graph of Compound 80 CH1-3/CT-tagged trastuzumabconjugate is 97.4% monomeric as determined by analytical SEC.

FIG. 116 shows a graph of Compound 80 CH1-3/CT-tagged sacituzumabconjugate yields a DAR of 6.19 as determined by PLRP.

FIG. 117 shows a graph of Compound 80 CH1-3/CT-tagged sacituzumabconjugate is 97.1% monomeric as determined by analytical SEC.

FIG. 118 shows a graph of Compound 86 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 5.46 as determined by PLRP.

FIG. 119 shows a graph of Compound 86 CH1-3/CT-tagged anti-FITCconjugate is 98.0% monomeric as determined by analytical SEC.

FIG. 120 shows a graph of Compound 92 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 1.58 as determined by PLRP.

FIG. 121 shows a graph of Compound 92 CH1-3/CT-tagged anti-FITCconjugate is 96.1% monomeric as determined by analytical SEC.

FIG. 122 shows a graph of Compound 99 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 3.07 as determined by PLRP.

FIG. 123 shows a graph of Compound 99 CH1-3/CT-tagged anti-FITCconjugate is 97.9% monomeric as determined by analytical SEC.

FIG. 124 shows a graph of Compound 103 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 6.56 as determined by PLRP.

FIG. 125 shows a graph of Compound 103 CH1-3/CT-tagged trastuzumabconjugate is 97.3% monomeric as determined by analytical SEC.

FIG. 126 shows a graph of Compound 110 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 5.66 as determined by PLRP.

FIG. 127 shows a graph of Compound 110 CH1-3/CT-tagged anti-FITCconjugate is 98.5% monomeric as determined by analytical SEC.

FIG. 128 shows a graph of Compound 113 CH1-3/CT-tagged sacituzumabconjugate yields a DAR of 6.41 as determined by PLRP.

FIG. 129 shows a graph of Compound 113 CH1-3/CT-tagged sacituzumabconjugate is 97.4% monomeric as determined by analytical SEC.

FIG. 130 shows a graph of Compound 123 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 5.56 as determined by PLRP.

FIG. 131 shows a graph of Compound 123 CH1-3/CT-tagged anti-FITCconjugate is 95.5% monomeric as determined by analytical SEC.

FIG. 132 shows a graph of Compound 151 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 5.67 as determined by PLRP.

FIG. 133 shows a graph of Compound 151 CH1-3/CT-tagged anti-FITCconjugate is 97.8% monomeric as determined by analytical SEC.

FIG. 134 shows a graph of Compound 147 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 6.47 as determined by PLRP.

FIG. 135 shows a graph of Compound 147 CH1-3/CT-tagged anti-FITCconjugate is 96.4% monomeric as determined by analytical SEC.

FIG. 136 shows a graph of Compound 73 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 5.41 as determined by PLRP.

FIG. 137 shows a graph of Compound 67 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 4.02 as determined by PLRP.

FIG. 138 shows a graph of Compound 136 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 7.26 as determined by PLRP.

FIG. 139 shows a graph of Compound 136 CH1-3/CT-tagged trastuzumabconjugate is 98.9% monomeric as determined by analytical SEC.

FIG. 140 shows a graph of Compound 142 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 6.9 as determined by PLRP.

FIG. 141 shows a graph of Compound 175 CH1-3/CT-tagged anti-FITCconjugate yields a DAR of 5.08 as determined by PLRP.

FIG. 142 shows a graph of Compound 175 CH1-3/CT-tagged anti-FITCconjugate is 93.0% monomeric as determined by analytical SEC.

FIG. 143 shows a graph of Compound 155 CH1-3/CT-tagged trastuzumabconjugate yields a DAR of 2.82 as determined by HIC.

FIG. 144 shows a graph of Compound 155 CH1-3/CT-tagged anti-FITCconjugate is 89.7% monomeric as determined by analytical SEC.

DEFINITIONS

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way ofexample, linear and branched hydrocarbyl groups such as methyl (CH₃—),ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl(CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—),t-butyl ((CH₃)₃C—), n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain (except the C₁carbon atom) have been optionally replaced with a heteroatom such as—O—, —N—, —S—, —S(O)_(n)— (where n is 0 to 2), —NR— (where R is hydrogenor alkyl) and having from 1 to 5 substituents selected from the groupconsisting of alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl, —SO₂-heteroaryl, and—NR^(a)R^(b), wherein R′ and R″ may be the same or different and arechosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

The term “alkane” refers to alkyl group and alkylene group, as definedherein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R′NHR″— where R′ is alkyl group as defined hereinand R″ is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O-, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group —NH-alkoxy, wherein alkoxy isdefined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “haloalkyl” refers to a substituted alkyl group as describedabove, wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl,trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

“Alkenyl” refers to straight chain or branched hydrocarbyl groups havingfrom 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and havingat least 1 and preferably from 1 to 2 sites of double bond unsaturation.This term includes, by way of example, bi-vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

The term “substituted alkenyl” refers to an alkenyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is asdefined herein. Alkynyloxy includes, by way of example, ethynyloxy,propynyloxy, and the like.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclyl-C(O)—, and substitutedheterocyclyl-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. For example, acylincludes the “acetyl” group CH₃C(O)—

“Acylamino” refers to the groups —NR²⁰C(O)alkyl, —NR²⁰C(O)substitutedalkyl, NR²⁰C(O)cycloalkyl, —NR²⁰C(O)substituted cycloalkyl,—NR²⁰C(O)cycloalkenyl, —NR²⁰C(O)substituted cycloalkenyl,—NR²⁰C(O)alkenyl, —NR²⁰C(O)substituted alkenyl, —NR²⁰C(O)alkynyl,—NR²⁰C(O)substituted alkynyl, —NR²⁰C(O)aryl, —NR²⁰C(O)substituted aryl,—NR²⁰C(O)heteroaryl, —NR²⁰C(O)substituted heteroaryl,—NR²⁰C(O)heterocyclic, and —NR²⁰C(O)substituted heterocyclic, whereinR²⁰ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyl” or the term “aminoacyl” refers to the group—C(O)NR²¹R²², wherein R²¹ and R²² independently are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R²¹ and R²² are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonylamino” refers to the group —NR²¹C(O)NR²²R²³ where R²¹,R²², and R²³ are independently selected from hydrogen, alkyl, aryl orcycloalkyl, or where two R groups are joined to form a heterocyclylgroup.

The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where eachR is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclyl are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclyl-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclyl are as defined herein.

“Aminosulfonyl” refers to the group —SO₂NR²¹R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic and where R²¹ and R²²are optionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group and alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 18 carbon atoms having a single ring (such as is present in aphenyl group) or a ring system having multiple condensed rings (examplesof such aromatic ring systems include naphthyl, anthryl and indanyl)which condensed rings may or may not be aromatic, provided that thepoint of attachment is through an atom of an aromatic ring. This termincludes, by way of example, phenyl and naphthyl. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, or from 1to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

“Aryloxy” refers to the group —O-aryl, wherein aryl is as definedherein, including, by way of example, phenoxy, naphthoxy, and the like,including optionally substituted aryl groups as also defined herein.

“Amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one R is not hydrogen.

The term “azido” refers to the group —N₃.

“Carboxyl,” “carboxy” or “carboxylate” refers to —CO₂H or salts thereof.

“Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or“carboxylalkyl” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl,—C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O-substituted aryl,—C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-cycloalkenyl,—C(O)O-substituted cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substitutedheteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted heterocyclic,wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“(Carboxyl ester)oxy” or “carbonate” refers to the groups—O—C(O)O-alkyl, —O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl,—O—C(O)O-substituted alkenyl, —O—C(O)O— alkynyl, —O—C(O)O-substitutedalkynyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O— cycloalkyl,—O—C(O)O-substituted cycloalkyl, —O—C(O)O-cycloalkenyl,—O—C(O)O-substituted cycloalkenyl, —O—C(O)O-heteroaryl,—O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclic, and—O—C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Cyano” or “nitrile” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyland the like. Such cycloalkyl groups include, by way of example, singlering structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclooctyl, and the like, or multiple ring structures such asadamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple rings and having at least onedouble bond and preferably from 1 to 2 double bonds.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10carbon atoms having single or multiple rings and having at least onetriple bond.

“Cycloalkoxy” refers to —O-cycloalkyl.

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms,such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected fromthe group consisting of oxygen, nitrogen, and sulfur within the ring.Such heteroaryl groups can have a single ring (such as, pyridinyl,imidazolyl or furyl) or multiple condensed rings in a ring system (forexample as in groups such as, indolizinyl, quinolinyl, benzofuran,benzimidazolyl or benzothienyl), wherein at least one ring within thering system is aromatic. To satisfy valence requirements, anyheteroatoms in such heteroaryl rings may or may not be bonded to H or asubstituent group, e.g., an alkyl group or other substituent asdescribed herein. In certain embodiments, the nitrogen and/or sulfurring atom(s) of the heteroaryl group are optionally oxidized to providefor the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This termincludes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl,and furanyl. Unless otherwise constrained by the definition for theheteroaryl substituent, such heteroaryl groups can be optionallysubstituted with 1 to 5 substituents, or from 1 to 3 substituents,selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substitutedalkoxy, substituted alkenyl, substituted alkynyl, substitutedcycloalkyl, substituted cycloalkenyl, amino, substituted amino,aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl, andtrihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. This term includes, by wayof example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

“Heteroaryloxy” refers to —O-heteroaryl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen,where, in fused ring systems, one or more of the rings can becycloalkyl, aryl, or heteroaryl, provided that the point of attachmentis through the non-aromatic ring. In certain embodiments, the nitrogenand/or sulfur atom(s) of the heterocyclic group are optionally oxidizedto provide for the N-oxide, —S(O)—, or —SO₂— moieties. To satisfyvalence requirements, any heteroatoms in such heterocyclic rings may ormay not be bonded to one or more H or one or more substituent group(s),e.g., an alkyl group or other substituent as described herein.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl, and fused heterocycle.

“Heterocyclyloxy” refers to the group —O-heterocyclyl.

The term “heterocyclylthio” refers to the group heterocyclic-S-.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein.

The term “hydroxyamino” refers to the group —NHOH.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

“Sulfonyl” refers to the group —SO₂-alkyl, —SO₂-substituted alkyl,—SO₂-alkenyl, —SO₂-substituted alkenyl, —SO₂-cycloalkyl,—SO₂-substituted cycloalkyl, —SO₂-cycloalkenyl, —SO₂-substitutedcylcoalkenyl, —SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl,—SO₂-substituted heteroaryl, —SO₂-heterocyclic, and —SO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein. Sulfonyl includes, by way ofexample, methyl-SO₂—, phenyl-SO₂—, and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group —OSO₂-alkyl, —OSO₂-substituted alkyl,—OSO₂-alkenyl, —OSO₂-substituted alkenyl, —OSO₂-cycloalkyl,—OSO₂-substituted cycloalkyl, —OSO₂-cycloalkenyl, —OSO₂-substitutedcylcoalkenyl, —OSO₂-aryl, —OSO₂-substituted aryl, —OSO₂-heteroaryl,—OSO₂-substituted heteroaryl, —OSO₂-heterocyclic, and —OSO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

“Sulfate” or “sulfate ester” refers the group —O—SO₂—OH, —O—SO₂—O-alkyl,—O—SO₂—O-substituted alkyl, —O—SO₂—O-alkenyl, —O—SO₂—O-substitutedalkenyl, —O—SO₂—O-cycloalkyl, —O—SO₂—O-substituted cycloalkyl,—O—SO₂—O-cycloalkenyl, —O-SO₂—O-substituted cylcoalkenyl, —O—SO₂—O-aryl,—O—SO₂—O-substituted aryl, —O—SO₂—O-heteroaryl, —O-SO₂—O-substitutedheteroaryl, —O—SO₂—O-heterocyclic, and —O—SO₂—O-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each Ris independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thioxo” or the term “thioketo” refers to the atom (═S).

“Alkylthio” or the term “thioalkoxy” refers to the group —S-alkyl,wherein alkyl is as defined herein. In certain embodiments, sulfur maybe oxidized to —S(O)—. The sulfoxide may exist as one or morestereoisomers.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined herein including optionally substituted aryl groupsalso defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined herein including optionallysubstituted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl-S—wherein the heterocyclyl group is as defined herein including optionallysubstituted heterocyclyl groups as also defined herein.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms of the specified group or radical are each, independentlyof one another, replaced with the same or different substituent groupsas defined below.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for substituting for one or more hydrogens(any two hydrogens on a single carbon can be replaced with ═O, ═NR⁷⁰,═N-OR⁷⁰, ═N₂ or ═S) on saturated carbon atoms in the specified group orradical are, unless otherwise specified, —R⁶⁰, halo, ═O, —OR⁷⁰, —SR⁷⁰,—NR⁸⁰R⁸⁰, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —SO₂R⁷⁰,—SO₂O⁻M⁺, —SO₂OR⁷⁰, —OSO₂R⁷⁰, —OSO₂O⁻M⁺, —OSO₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂,—P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—C(O)O⁻M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰,—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O)O⁻M⁺, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰,—NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ isselected from the group consisting of optionally substituted alkyl,cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each R⁷⁰ is independentlyhydrogen or R⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, twoR⁸⁰'s, taken together with the nitrogen atom to which they are bonded,form a 5-, 6- or 7-membered heterocycloalkyl which may optionallyinclude from 1 to 4 of the same or different additional heteroatomsselected from the group consisting of O, N and S, of which N may have —Hor C₁-C₃ alkyl substitution; and each M⁺is a counter ion with a netsingle positive charge. Each M⁺may independently be, for example, analkali ion, such as K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; oran alkaline earth ion, such as [Ca²⁺]_(0.5), [Mg²⁺]_(0.5), or[Ba²⁺]_(0.5) (“subscript 0.5 means that one of the counter ions for suchdivalent alkali earth ions can be an ionized form of a compound of theinvention and the other a typical counter ion such as chloride, or twoionized compounds disclosed herein can serve as counter ions for suchdivalent alkali earth ions, or a doubly ionized compound of theinvention can serve as the counter ion for such divalent alkali earthions). As specific examples, —NR⁸⁰R⁸⁰ is meant to include —NH₂,—NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl andN-morpholinyl.

In addition to the disclosure herein, substituent groups for hydrogenson unsaturated carbon atoms in “substituted” alkene, alkyne, aryl andheteroaryl groups are, unless otherwise specified, —R⁶⁰, halo, —O⁻M⁺,—OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰, trihalomethyl, —CF₃, —CN, —OCN, —SCN,—NO, —NO₂, —N₃, —SO₂R⁷⁰, —SO₃M⁺, —SO₃R⁷⁰, —OSO₂R⁷⁰, —OSO₃ ⁻M⁺, —OSO₃R⁷⁰,—PO₃ ⁻²(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰,—C(NR⁷⁰)R⁷⁰, —CO₂ ⁻M⁺, —CO₂R⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰,—C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰, —OCO₂ ⁻M⁺, —OCO₂R⁷⁰, —OC(S)OR⁷⁰,—NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰,—NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰,R⁷⁰, R⁸⁰ and M⁺are as previously defined, provided that in case ofsubstituted alkene or alkyne, the substituents are not —O⁻M⁺, —OR⁷⁰,—SR⁷⁰, or —S⁻M⁺.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for hydrogens on nitrogen atoms in“substituted” heteroalkyl and cycloheteroalkyl groups are, unlessotherwise specified, —R⁶⁰, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰,trihalomethyl, —CF₃, —CN, —NO, —NO₂, —S(O)₂R⁷⁰, —S(O)₂O⁻M⁺, —S(O)₂OR⁷⁰,—OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺,—P(O)(OR⁷⁰)(OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —C(O)OR⁷⁰,—C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰,—OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)OR⁷⁰,—NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺are as previouslydefined.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (salts withcounterions having acceptable mammalian safety for a given dosageregime). Such salts can be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart and include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, formate, tartrate, besylate,mesylate, acetate, maleate, oxalate, and the like.

The term “salt thereof” means a compound formed when a proton of an acidis replaced by a cation, such as a metal cation or an organic cation andthe like. Where applicable, the salt is a pharmaceutically acceptablesalt, although this is not required for salts of intermediate compoundsthat are not intended for administration to a patient. By way ofexample, salts of the present compounds include those wherein thecompound is protonated by an inorganic or organic acid to form a cation,with the conjugate base of the inorganic or organic acid as the anioniccomponent of the salt.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric ring atom arrangements are possible.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of subject compound.

“Pharmaceutically effective amount” and “therapeutically effectiveamount” refer to an amount of a compound sufficient to treat a specifieddisorder or disease or one or more of its symptoms and/or to prevent theoccurrence of the disease or disorder. In reference to tumorigenicproliferative disorders, a pharmaceutically or therapeutically effectiveamount comprises an amount sufficient to, among other things, cause thetumor to shrink or decrease the growth rate of the tumor.

“Patient” refers to human and non-human subjects, especially mammaliansubjects.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition in a patient, such as amammal (particularly a human) that includes: (a) preventing the diseaseor medical condition from occurring, such as, prophylactic treatment ofa subject; (b) ameliorating the disease or medical condition, such as,eliminating or causing regression of the disease or medical condition ina patient; (c) suppressing the disease or medical condition, for exampleby, slowing or arresting the development of the disease or medicalcondition in a patient; or (d) alleviating a symptom of the disease ormedical condition in a patient.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymeric form of amino acids ofany length. Unless specifically indicated otherwise, “polypeptide,”“peptide,” and “protein” can include genetically coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones. The termincludes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, proteins which contain at least oneN-terminal methionine residue (e.g., to facilitate production in arecombinant host cell); immunologically tagged proteins; and the like.In certain embodiments, a polypeptide is an antibody.

“Native amino acid sequence” or “parent amino acid sequence” are usedinterchangeably herein to refer to the amino acid sequence of apolypeptide prior to modification to include at least one modified aminoacid residue.

The terms “amino acid analog,” “unnatural amino acid,” and the like maybe used interchangeably, and include amino acid-like compounds that aresimilar in structure and/or overall shape to one or more amino acidscommonly found in naturally occurring proteins (e.g., Ala or A, Cys orC, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K,Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S,Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also includenatural amino acids with modified side chains or backbones. Amino acidanalogs also include amino acid analogs with the same stereochemistry asin the naturally occurring D-form, as well as the L-form of amino acidanalogs. In some instances, the amino acid analogs share backbonestructures, and/or the side chain structures of one or more naturalamino acids, with difference(s) being one or more modified groups in themolecule. Such modification may include, but is not limited to,substitution of an atom (such as N) for a related atom (such as S),addition of a group (such as methyl, or hydroxyl, etc.) or an atom (suchas Cl or Br, etc.), deletion of a group, substitution of a covalent bond(single bond for double bond, etc.), or combinations thereof. Forexample, amino acid analogs may include α-hydroxy acids, and α-aminoacids, and the like. Examples of amino acid analogs include, but are notlimited to, sulfoalanine, and the like.

The terms “amino acid side chain” or “side chain of an amino acid” andthe like may be used to refer to the substituent attached to theα-carbon of an amino acid residue, including natural amino acids,unnatural amino acids, and amino acid analogs. An amino acid side chaincan also include an amino acid side chain as described in the context ofthe modified amino acids and/or conjugates described herein.

The term “carbohydrate” and the like may be used to refer to monomersunits and/or polymers of monosaccharides, disaccharides,oligosaccharides, and polysaccharides. The term sugar may be used torefer to the smaller carbohydrates, such as monosaccharides,disaccharides. The term “carbohydrate derivative” includes compoundswhere one or more functional groups of a carbohydrate of interest aresubstituted (replaced by any convenient substituent), modified(converted to another group using any convenient chemistry) or absent(e.g., eliminated or replaced by H). A variety of carbohydrates andcarbohydrate derivatives are available and may be adapted for use in thesubject compounds and conjugates.

The term “glycoside” or “glycosyl” refers to a sugar molecule or groupbound to a moiety via a glycosidic bond. For example, the moiety thatthe glycoside is bound to can be a cleavable linker as described herein.A glycosidic bond can link the glycoside to the other moiety throughvarious types of bonds, such as, but not limited to, an O-glycosidicbond (an O-glycoside), an N-glycosidic bond (a glycosylamine), anS-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycosideor C-glycosyl). In some cases, glycosides can be cleaved from the moietythey are attached to, such as by chemically-mediated hydrolysis orenzymatically-mediated hydrolysis.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, and multispecific antibodies (e.g., bispecificantibodies), humanized antibodies, single-chain antibodies, chimericantibodies, antibody fragments (e.g., Fab fragments), and the like. Anantibody is capable of binding a target antigen. (Janeway, C., Travers,P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., GarlandPublishing, New York). A target antigen can have one or more bindingsites, also called epitopes, recognized by complementarity determiningregions (CDRs) formed by one or more variable regions of an antibody.

The term “natural antibody” refers to an antibody in which the heavy andlight chains of the antibody have been made and paired by the immunesystem of a multi-cellular organism. Spleen, lymph nodes, bone marrowand serum are examples of tissues that produce natural antibodies. Forexample, the antibodies produced by the antibody producing cellsisolated from a first animal immunized with an antigen are naturalantibodies.

The term “humanized antibody” or “humanized immunoglobulin” refers to anon-human (e.g., mouse or rabbit) antibody containing one or more aminoacids (in a framework region, a constant region or a CDR, for example)that have been substituted with a correspondingly positioned amino acidfrom a human antibody. In general, humanized antibodies produce areduced immune response in a human host, as compared to a non-humanizedversion of the same antibody. Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). In certain embodiments, framework substitutions areidentified by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann etal., Nature 332:323 (1988)). Additional methods for humanizingantibodies contemplated for use in the present invention are describedin U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417;5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO98/45331 and WO 98/45332. In particular embodiments, a subject rabbitantibody may be humanized according to the methods set forth inUS20040086979 and US20050033031. Accordingly, the antibodies describedabove may be humanized using methods that are well known in the art.

The term “chimeric antibodies” refer to antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. An example of a therapeutic chimeric antibody is ahybrid protein composed of the variable or antigen-binding domain from amouse antibody and the constant or effector domain from a humanantibody, although domains from other mammalian species may be used.

An immunoglobulin polypeptide immunoglobulin light or heavy chainvariable region is composed of a framework region (FR) interrupted bythree hypervariable regions, also called “complementarity determiningregions” or “CDRs”. The extent of the framework region and CDRs havebeen defined (see, “Sequences of Proteins of Immunological Interest,” E.Kabat et al., U.S. Department of Health and Human Services, 1991). Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs. The CDRs are primarily responsible for binding to an epitopeof an antigen.

A “parent Ig polypeptide” is a polypeptide comprising an amino acidsequence which lacks an aldehyde-tagged constant region as describedherein. The parent polypeptide may comprise a native sequence constantregion, or may comprise a constant region with pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions).

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,at least 75% free, at least 80% free, at least 85% free, at least 90%free, at least 95% free, at least 98% free, or more than 98% free, fromother components with which it is naturally associated.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

By “reactive partner” is meant a molecule or molecular moiety thatspecifically reacts with another reactive partner to produce a reactionproduct. Exemplary reactive partners include a cysteine or serine of asulfatase motif and Formylglycine Generating Enzyme (FGE), which reactto form a reaction product of a converted aldehyde tag containing aformylglycine (fGly) in lieu of cysteine or serine in the motif. Otherexemplary reactive partners include an aldehyde of an fGly residue of aconverted aldehyde tag (e.g., a reactive aldehyde group) and an“aldehyde-reactive reactive partner”, which comprises analdehyde-reactive group and a moiety of interest, and which reacts toform a reaction product of a modified aldehyde tagged polypeptide havingthe moiety of interest conjugated to the modified polypeptide through athe fGly residue.

“N-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free amine group, which amine group in non-N-terminus aminoacid residues normally forms part of the covalent backbone of thepolypeptide.

“C-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free carboxyl group, which carboxyl group in non-C-terminusamino acid residues normally forms part of the covalent backbone of thepolypeptide.

By “internal site” as used in referenced to a polypeptide or an aminoacid sequence of a polypeptide means a region of the polypeptide that isnot at the N-terminus or at the C-terminus.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace subject matter that are, for example, compoundsthat are stable compounds (i.e., compounds that can be made, isolated,characterized, and tested for biological activity). In addition, allsub-combinations of the various embodiments and elements thereof (e.g.,elements of the chemical groups listed in the embodiments describingsuch variables) are also specifically embraced by the present inventionand are disclosed herein just as if each and every such sub-combinationwas individually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Detailed Description

The present disclosure provides antibody-drug conjugate (ADC)structures, which include a camptothecine or a camptothecine derivativelinked to a polypeptide (e.g., an antibody) through a linker. Thedisclosure also encompasses compounds and methods for production of suchconjugates, as well as methods of using the conjugates.

Antibody-Drug Conjugates

The present disclosure provides a conjugate, e.g., an antibody-drugconjugate (ADC). By “conjugate” is meant a polypeptide (e.g., anantibody) is covalently attached to one or more other moieties (e.g.,drugs or active agents). For example, an antibody-drug conjugateaccording to the present disclosure includes one or more drugs or activeagents covalently attached to an antibody. In certain embodiments, thepolypeptide (e.g., antibody) and the one or more drugs or active agentsare bound to each other through one or more functional groups andcovalent bonds. For example, the one or more functional groups andcovalent bonds can include a cleavable linker as described herein.

In certain embodiments, the conjugate is a polypeptide conjugate, whichincludes a polypeptide (e.g., an antibody) conjugated to one or moreother moieties. In certain embodiments, the one or more moietiesconjugated to the polypeptide can each independently be any of a varietyof moieties of interest such as, but not limited to, a drug, an activeagent, a detectable label, a water-soluble polymer, or a moiety forimmobilization of the polypeptide to a membrane or a surface. In certainembodiments, the conjugate is a drug conjugate, where a polypeptide isan antibody, thus providing an antibody-drug conjugate. For instance,the conjugate can be a drug conjugate, where a polypeptide is conjugatedto one or more drugs or active agents. In certain embodiments the drugor active agent is a camptothecine or a camptothecine derivative.Various types of camptothecine or camptothecine derivatives may be usedin the conjugates and are described in more detail below.

The one or more drugs or active agents can be conjugated to thepolypeptide (e.g., antibody) at any desired site of the polypeptide.Thus, the present disclosure provides, for example, a polypeptide havinga drug or active agent conjugated at a site at or near the C-terminus ofthe polypeptide. Other examples include a polypeptide having a drug oractive agent conjugated at a position at or near the N-terminus of thepolypeptide. Examples also include a polypeptide having a drug or activeagent conjugated at a position between the C-terminus and the N-terminusof the polypeptide (e.g., at an internal site of the polypeptide).Combinations of the above are also possible where the polypeptide isconjugated to two or more drugs or active agents.

In certain embodiments, a conjugate of the present disclosure includesone or more drugs or active agents conjugated to an amino acid residueof a polypeptide at the α-carbon of an amino acid residue. Statedanother way, a conjugate includes a polypeptide where the side chain ofone or more amino acid residues in the polypeptide has been modified andattached to one or more drugs or active agents (e.g., attached to one ormore drugs or active agents through a linker as described herein). Forexample, a conjugate includes a polypeptide where the α-carbon of one ormore amino acid residues in the polypeptide has been modified andattached to one or more drugs or active agents (e.g., attached to one ormore drugs or active agents through a linker as described herein).

Embodiments of the present disclosure include conjugates where apolypeptide is conjugated to one or more moieties, such as 2 moieties, 3moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9moieties, or 10 or more moieties. The moieties may be conjugated to thepolypeptide at one or more sites in the polypeptide. For example, one ormore moieties may be conjugated to a single amino acid residue of thepolypeptide. In some cases, one moiety is conjugated to an amino acidresidue of the polypeptide. In other embodiments, two moieties may beconjugated to the same amino acid residue of the polypeptide. In otherembodiments, a first moiety is conjugated to a first amino acid residueof the polypeptide and a second moiety is conjugated to a second aminoacid residue of the polypeptide. Combinations of the above are alsopossible, for example where a polypeptide is conjugated to a firstmoiety at a first amino acid residue and conjugated to two othermoieties at a second amino acid residue. Other combinations are alsopossible, such as, but not limited to, a polypeptide conjugated to firstand second moieties at a first amino acid residue and conjugated tothird and fourth moieties at a second amino acid residue, etc. In somecases, two or more amino acid residues in the polypeptide are eachconjugated to a pair of moieties (i.e., two moieties), where each pairof moieties is conjugated to the polypeptide through a branched linkeras described herein. In some cases, 1 amino acid residue in thepolypeptide is conjugated to a pair of moieties through a branchedlinker as described herein.

The one or more amino acid residues of the polypeptide that areconjugated to the one or more moieties may be naturally occurring aminoacids, unnatural amino acids, or combinations thereof. For instance, theconjugate may include one or more drugs or active agents conjugated to anaturally occurring amino acid residue of the polypeptide. In otherinstances, the conjugate may include one or more drugs or active agentsconjugated to an unnatural amino acid residue of the polypeptide. One ormore drugs or active agents may be conjugated to the polypeptide at asingle natural or unnatural amino acid residue as described above. Oneor more natural or unnatural amino acid residues in the polypeptide maybe conjugated to the moiety or moieties as described herein. Forexample, two (or more) amino acid residues (e.g., natural or unnaturalamino acid residues) in the polypeptide may each be conjugated to one ortwo moieties, such that multiple sites in the polypeptide are conjugatedto the moieties of interest.

In certain embodiments, the polypeptide (e.g., antibody) and the moietyof interest (e.g., drug or active agent) are conjugated through aconjugation moiety. For example, the polypeptide and the moiety ofinterest may each be bound (e.g., covalently bonded) to the conjugationmoiety, thus indirectly binding the polypeptide and the moiety ofinterest together through the conjugation moiety. In some cases, theconjugation moiety includes a hydrazinyl-indolyl or ahydrazinyl-pyrrolo-pyridinyl compound, or a derivative of ahydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound. Forinstance, a general scheme for coupling a moiety of interest to apolypeptide through a hydrazinyl-indolyl or ahydrazinyl-pyrrolo-pyridinyl conjugation moiety is shown in the generalreaction scheme below. Hydrazinyl-indolyl andhydrazinyl-pyrrolo-pyridinyl conjugation moiety are also referred toherein as a hydrazino-iso-Pictet-Spengler (HIPS) conjugation moiety andan aza-hydrazino-iso-Pictet-Spengler (azaHIPS) conjugation moiety,respectively.

In the reaction scheme above, R includes the moiety of interest (e.g., adrug or active agent) that is conjugated to the polypeptide (e.g.,conjugated to the polypeptide through a cleavable linker as describedherein). As shown in the reaction scheme above, a polypeptide thatincludes a 2-formylglycine residue (fGly) is reacted with a drug oractive agent that has been modified to include a conjugation moiety(e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinylconjugation moiety) to produce a polypeptide conjugate attached to theconjugation moiety, thus attaching the drug or active agent to thepolypeptide through the conjugation moiety.

As described herein, the moiety can be any of a variety of moieties suchas, but not limited to, chemical entities, such as detectable labels, ordrugs or active agents. R′ and R″ may each independently be any desiredsubstituent, such as, but not limited to, hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Zmay be CR²¹, NR²², N, O or S, where R²¹ and R²² are each independentlyselected from any of the substituents described for R′ and R″ above.

Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl couplingmoieties are also possible, as shown in the conjugates and compoundsdescribed herein. For example, the hydrazinyl-indolyl orhydrazinyl-pyrrolo-pyridinyl coupling moieties may be attached (e.g.,covalently attached) to a linker. As such, embodiments of the presentdisclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinylconjugation moiety attached to a drug or active agent through a linker.Various embodiments of the linker that may couple the hydrazinyl-indolylor hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the drug or activeagent are described in detail herein. For example, in some instances,the linker is a cleavable linker, such as a cleavable linker asdescribed herein.

In some instances, the hydrazinyl-indolyl orhydrazinyl-pyrrolo-pyridinyl conjugation moieties may be attached (e.g.,covalently attached) to two or more linkers. As such, embodiments of thepresent disclosure include a hydrazinyl-indolyl orhydrazinyl-pyrrolo-pyridinyl conjugation moiety attached to two or moredrugs or active agents each through a corresponding linker. Thus,conjugates of the present disclosure may include two or more linkers,where each linker attaches a corresponding drug or active agent to thehydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.Accordingly, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinylconjugation moiety and two or more linkers may be viewed overall as a“branched linker”, where the hydrazinyl-indolyl orhydrazinyl-pyrrolo-pyridinyl conjugation moiety is attached to two ofmore “branches”, where each branch includes a linker attached to a drugor active agent.

In certain embodiments, the polypeptide may be conjugated to one or moremoieties of interest, where one or more amino acid residues of thepolypeptide are modified before conjugation to the moiety of interest.Modification of one or more amino acid residues of the polypeptide mayproduce a polypeptide that contains one or more reactive groups suitablefor conjugation to the moiety of interest. In some cases, thepolypeptide may be include one or more modified amino acid residues toprovide one or more reactive groups suitable for conjugation to themoiety of interest (e.g., one or more moieties that includes aconjugation moiety, such as a hydrazinyl-indolyl or ahydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above). Forexample, an amino acid of the polypeptide may be modified to include areactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehydemay be included in an “aldehyde tag” or “ald-tag”, which as used hereinrefers to an amino acid sequence derived from a sulfatase motif (e.g.,L(C/S)TPSR) that has been converted by action of a formylglycinegenerating enzyme (FGE) to contain a 2-formylglycine residue (referredto herein as “fGly”). The fGly residue generated by an FGE may also bereferred to as a “formylglycine”. Stated differently, the term “aldehydetag” is used herein to refer to an amino acid sequence that includes a“converted” sulfatase motif (i.e., a sulfatase motif in which a cysteineor serine residue has been converted to fGly by action of an FGE, e.g.,L(fGly)TPSR). A converted sulfatase motif may be produced from an aminoacid sequence that includes an “unconverted” sulfatase motif (i.e., asulfatase motif in which the cysteine or serine residue has not beenconverted to fGly by an FGE, but is capable of being converted, e.g., anunconverted sulfatase motif with the sequence: L(C/S)TPSR). By“conversion” as used in the context of action of a formylglycinegenerating enzyme (FGE) on a sulfatase motif refers to biochemicalmodification of a cysteine or serine residue in a sulfatase motif to aformylglycine (fGly) residue (e.g., Cys to fGly, or Ser to fGly).Additional aspects of aldehyde tags and uses thereof in site-specificprotein modification are described in U.S. Pat. Nos. 7,985,783 and8,729,232, the disclosures of each of which are incorporated herein byreference.

In some cases, to produce the conjugate, the polypeptide containing thefGly residue may be conjugated to the moiety of interest by reaction ofthe fGly with a compound (e.g., a compound containing ahydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety,as described above). For example, an fGly-containing polypeptide may becontacted with a reactive partner-containing drug under conditionssuitable to provide for conjugation of the drug to the polypeptide. Insome instances, the reactive partner-containing drug may include ahydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moietyas described above. For example, a drug or active agent may be modifiedto include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinylconjugation moiety. In some cases, the drug or active agent is attachedto a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl, such ascovalently attached to a hydrazinyl-indolyl or ahydrazinyl-pyrrolo-pyridinyl through a linker, such as a linker asdescribed in detail herein.

In certain embodiments, a conjugate of the present disclosure includes apolypeptide (e.g., an antibody) having at least one amino acid residuethat has been attached to one or more moieties of interest (e.g., drugsor active agents). In order to make the conjugate, an amino acid residueof the polypeptide may be modified and then coupled to one or more drugsor active agents attached to a hydrazinyl-indolyl or ahydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above. Incertain embodiments, an amino acid residue of the polypeptide (e.g.,antibody) is a cysteine or serine residue that is modified to an fGlyresidue, as described above. In certain embodiments, the modified aminoacid residue (e.g. fGly residue) is conjugated to a drug or active agentcontaining a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinylconjugation moiety as described above to provide a conjugate of thepresent disclosure where the one or more drugs or active agents areconjugated to the polypeptide through the hydrazinyl-indolyl orhydrazinyl-pyrrolo-pyridinyl conjugation moiety. As used herein, theterm fGly′ refers to the modified amino acid residue of the polypeptide(e.g., antibody) that is coupled to the moiety of interest (e.g., a drugor active agent).

In certain embodiments, the conjugate includes a polypeptide (e.g. anantibody) having at least one amino acid residue attached to a linker asdescribed herein, which in turn is attached to one or more drugs oractive agents. For instance, the conjugate may include a polypeptide(e.g., an antibody) having at least one amino acid residue (fGly′) thatis conjugated to the one or more moieties of interest (e.g., one or moredrugs or active agents) as described above.

Aspects of the present disclosure include a conjugate of formula (I):

wherein

Z is CR¹⁰ or N,

R⁷ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R⁸ and R⁹ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁸ and R⁹ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

each R¹⁰ is independently selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

W is a polypeptide;

L is a linker attached to a compound of formula (II) at R¹, R², R³, R⁴,R⁵ or R⁶:

R¹ and R² are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R¹ and R² are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R³ and R⁴ are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R³ and R⁴ are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R⁵ is selected from hydrogen, halogen, hydroxy, amino, substitutedamino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

R⁶ is selected from OH and OC(O)R¹¹; and

R¹¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl, wherein at leastone R¹⁰ is optionally linked to a second compound of formula (II).

The substituents related to conjugates of formula (I) are described inmore detail below.

In certain embodiments, Z is CR¹⁰ or N. In certain embodiments, Z isCR¹⁰. In certain embodiments, Z is N.

In certain embodiments, R⁷ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁷ is hydrogen. In certain embodiments, R⁷ is alkylor substituted alkyl, such as C₁₋₆ alkyl or C₁₋₆ substituted alkyl, orC₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃ alkyl or C₁₋₃ substitutedalkyl. In certain embodiments, R⁷ is methyl. In certain embodiments, R⁷is alkenyl or substituted alkenyl, such as C₂₋₆ alkenyl or C₂₋₆substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, orC₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certain embodiments, R⁷ isalkynyl or substituted alkynyl, such as C₂₋₆ alkenyl or C₂₋₆ substitutedalkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, or C₂₋₃ alkenyl orC₂₋₃ substituted alkenyl. In certain embodiments, R⁷ is aryl orsubstituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl, such as aC₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substituted aryl. Incertain embodiments, R⁷ is heteroaryl or substituted heteroaryl, such asC₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, such as a C₅ heteroarylor C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆ substitutedheteroaryl. In certain embodiments, R⁷ is cycloalkyl or substitutedcycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substituted cycloalkyl, suchas a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, or a C₃₋₅cycloalkyl or C₃₋₅ substituted cycloalkyl. In certain embodiments, R⁷ isheterocyclyl or substituted heterocyclyl, such as C₃₋₈ heterocyclyl orC₃₋₈ substituted heterocyclyl, such as a C₃₋₆ heterocyclyl or C₃₋₆substituted heterocyclyl, or a C₃₋₅ heterocyclyl or C₃₋₅ substitutedheterocyclyl.

In certain embodiments, R⁸ and R⁹ are each independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, or R⁸ and R⁹ are optionally cyclically linkedto form a 5 or 6-membered heterocyclyl.

In certain embodiments, R⁸ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁸ is hydrogen. In certain embodiments, R⁸ is alkylor substituted alkyl, such as C₁₋₆ alkyl or C₁₋₆ substituted alkyl, orC₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃ alkyl or C₁₋₃ substitutedalkyl. In certain embodiments, R⁸ is methyl. In certain embodiments, R⁸is alkenyl or substituted alkenyl, such as C₂₋₆ alkenyl or C₂₋₆substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, orC₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certain embodiments, R⁸ isalkynyl or substituted alkynyl. In certain embodiments, R⁸ is alkoxy orsubstituted alkoxy. In certain embodiments, R⁸ is amino or substitutedamino. In certain embodiments, R⁸ is carboxyl or carboxyl ester. Incertain embodiments, R⁸ is acyl or acyloxy. In certain embodiments, R⁸is acyl amino or amino acyl. In certain embodiments, R⁸ is alkylamide orsubstituted alkylamide. In certain embodiments, R⁸ is sulfonyl. Incertain embodiments, R⁸ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R⁸ is aryl or substituted aryl, such as C₅₋₈ arylor C₅₋₈ substituted aryl, such as a C₅ aryl or C₅ substituted aryl, or aC₆ aryl or C₆ substituted aryl. In certain embodiments, R⁸ is heteroarylor substituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substitutedheteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or aC₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R⁸is cycloalkyl or substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈substituted cycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substitutedcycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. Incertain embodiments, R⁸ is heterocyclyl or substituted heterocyclyl,such as a C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R⁹ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁹ is hydrogen. In certain embodiments, R⁹ is alkylor substituted alkyl, such as C₁₋₆ alkyl or C₁₋₆ substituted alkyl, orC₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃ alkyl or C₁₋₃ substitutedalkyl. In certain embodiments, R⁹ is methyl. In certain embodiments, R⁹is alkenyl or substituted alkenyl, such as C₂₋₆ alkenyl or C₂₋₆substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, orC₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certain embodiments, R⁹ isalkynyl or substituted alkynyl. In certain embodiments, R⁹ is alkoxy orsubstituted alkoxy. In certain embodiments, R⁹ is amino or substitutedamino. In certain embodiments, R⁹ is carboxyl or carboxyl ester. Incertain embodiments, R⁹ is acyl or acyloxy. In certain embodiments, R⁹is acyl amino or amino acyl. In certain embodiments, R⁹ is alkylamide orsubstituted alkylamide. In certain embodiments, R⁹ is sulfonyl. Incertain embodiments, R⁹ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R⁹ is aryl or substituted aryl, such as C₅₋₈ arylor C₅₋₈ substituted aryl, such as a C₅ aryl or C₅ substituted aryl, or aC₆ aryl or C₆ substituted aryl. In certain embodiments, R⁹ is heteroarylor substituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substitutedheteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or aC₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R⁹is cycloalkyl or substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈substituted cycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substitutedcycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. Incertain embodiments, R⁹ is heterocyclyl or substituted heterocyclyl,such as C₃₋₈ heterocyclyl or C₃₋₈ substituted heterocyclyl, such as aC₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R⁸ and R⁹ are optionally cyclically linked toform a 5 or 6-membered heterocyclyl. In certain embodiments, R⁸ and R⁹are cyclically linked to form a 5 or 6-membered heterocyclyl. In certainembodiments, R⁸ and R⁹ are cyclically linked to form a 5-memberedheterocyclyl. In certain embodiments, R⁸ and R⁹ are cyclically linked toform a 6-membered heterocyclyl.

In certain embodiments, each R¹⁰ is independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

The various possibilities for each R¹⁰ are described in more detail asfollows. In certain embodiments, R¹⁰ is hydrogen. In certainembodiments, each R¹⁰ is hydrogen. In certain embodiments, R¹⁰ ishalogen, such as F, Cl, Br or I. In certain embodiments, R¹⁰ is F. Incertain embodiments, R¹⁰ is Cl. In certain embodiments, R¹⁰ is Br. Incertain embodiments, R¹⁰ is I. In certain embodiments, R¹⁰ is alkyl orsubstituted alkyl, such as C₁₋₆ alkyl or C₁₋₆ substituted alkyl, or C₁₋₄alkyl or C₁₋₄ substituted alkyl, or C₁₋₃ alkyl or C₁₋₃ substitutedalkyl. In certain embodiments, R¹⁰ is methyl. In certain embodiments,R¹⁰ is alkenyl or substituted alkenyl, such as C₂₋₆ alkenyl or C₂₋₆substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, orC₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certain embodiments, R¹⁰ isalkynyl or substituted alkynyl. In certain embodiments, R¹⁰ is alkoxy orsubstituted alkoxy. In certain embodiments, R¹⁰ is amino or substitutedamino. In certain embodiments, R¹⁰ is carboxyl or carboxyl ester. Incertain embodiments, R¹⁰ is acyl or acyloxy. In certain embodiments, R¹⁰is acyl amino or amino acyl. In certain embodiments, R¹⁰ is alkylamideor substituted alkylamide. In certain embodiments, R¹⁰ is sulfonyl. Incertain embodiments, R¹⁰ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R¹⁰ is aryl or substituted aryl, such as C₅₋₈ arylor C₅₋₈ substituted aryl, such as a C₅ aryl or C₅ substituted aryl, or aC₆ aryl or C₆ substituted aryl (e.g., phenyl or substituted phenyl). Incertain embodiments, R¹⁰ is heteroaryl or substituted heteroaryl, suchas C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, such as a C₅heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆substituted heteroaryl. In certain embodiments, R¹⁰ is cycloalkyl orsubstituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R¹⁰ is heterocyclyl or substituted heterocyclyl, such asC₃₋₈ heterocyclyl or C₃₋₈ substituted heterocyclyl, such as a C₃₋₆heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅ heterocyclyl orC₃₋₅ substituted heterocyclyl.

In certain embodiments, W is a polypeptide. For example, W can be anantibody. In certain embodiments, W comprises one or more fGly′ residuesas described herein. In certain embodiments, the polypeptide is attachedto the rest of the conjugate through an fGly′ residue as describedherein. Further description of polypeptides and antibodies that find usein the subject conjugates is found in the disclosure herein.

In certain embodiments, L is a linker attached to a compound of formula(II) at R¹, R², R³, R⁴, R⁵ or R⁶. Linkers suitable for L are describedin more detail below.

In certain embodiments, the conjugate of formula (I) includes a linker,L. The linker may be utilized to bind one or more moieties of interest(e.g., drug or active agent) to one or more polypeptides through aconjugation moiety. The linker may be bound (e.g., covalently bonded) tothe conjugation moiety (e.g., as described herein) at any convenientposition. For example, the linker may attach a hydrazinyl-indolyl or ahydrazinyl-pyrrolo-pyridinyl conjugation moiety to a drug (e.g., acamptothecine or camptothecine derivative). The hydrazinyl-indolyl orhydrazinyl-pyrrolo-pyridinyl conjugation moiety may be used to conjugatethe linker (and thus the drug) to a polypeptide, such as an antibody.For example, the conjugation moiety may be used to conjugate the linker(and thus the drug) to a modified amino acid residue of the polypeptide,such as an fGly reside of an antibody, as described herein.

For example, as shown in formula (I) above, L is attached to W through aconjugation moiety, and thus W is indirectly bonded to the linker Lthrough the conjugation moiety. As described above, W is a polypeptide(e.g., an antibody), and thus L is attached through the conjugationmoiety to the polypeptide (antibody), e.g., the linker L is indirectlybonded to the polypeptide (antibody) through the conjugation moiety(e.g., through a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinylconjugation moiety as described herein).

Any convenient linker may be utilized for the linker L in the subjectconjugates and compounds. In certain embodiments, the linker L mayinclude a group selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino,alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, thelinker L may include an alkyl or substituted alkyl group. In certainembodiments, the linker L may include an alkenyl or substituted alkenylgroup. In certain embodiments, the linker L may include an alkynyl orsubstituted alkynyl group. In certain embodiments, the linker L mayinclude an alkoxy or substituted alkoxy group. In certain embodiments,the linker L may include an amino or substituted amino group. In certainembodiments, the linker L may include a carboxyl or carboxyl estergroup. In certain embodiments, the linker L may include an acyl aminogroup. In certain embodiments, the linker L may include an alkylamide orsubstituted alkylamide group. In certain embodiments, the linker L mayinclude an aryl or substituted aryl group. In certain embodiments, thelinker L may include a heteroaryl or substituted heteroaryl group. Incertain embodiments, the linker L may include a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, the linker L mayinclude a heterocyclyl or substituted heterocyclyl group.

In certain embodiments, the linker L may include a polymer. For example,the polymer may include a polyalkylene glycol and derivatives thereof,including polyethylene glycol, methoxypolyethylene glycol, polyethyleneglycol homopolymers, polypropylene glycol homopolymers, copolymers ofethylene glycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol. Other linkers are also possible, as shown in theconjugates and compounds described in more detail below.

In some embodiments, L is a linker (e.g., a first linker) described bythe formula:

-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-(L⁶)_(f)-,

wherein L¹, L², L³, L⁴, L⁵ and L⁶ are each independently a linkersubunit, and a, b, c, d, e and f are each independently 0 or 1, whereinthe sum of a, b, c, d, e and f is 1 to 6.

In certain embodiments, the sum of a, b, c, d, e and f is 1. In certainembodiments, the sum of a, b, c, d, e and f is 2. In certainembodiments, the sum of a, b, c, d, e and f is 3. In certainembodiments, the sum of a, b, c, d, e and f is 4. In certainembodiments, the sum of a, b, c, d, e and f is 5. In certainembodiments, the sum of a, b, c, d, e and f is 6. In certainembodiments, a, b, c, d, e and f are each 1. In certain embodiments, a,b, c, d and e are each 1 and f is 0. In certain embodiments, a, b, c andd are each 1 and e and f are each 0. In certain embodiments, a, b, and care each 1 and d, e and f are each 0. In certain embodiments, a and bare each 1 and c, d, e and f are each 0. In certain embodiments, a is 1and b, c, d, e and f are each 0.

In certain embodiments, the linker subunit L¹ is attached to thehydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl conjugationmoiety (e.g., as shown in formula (I) above). In certain embodiments,the linker subunit L², if present, is attached to the camptothecine orcamptothecine derivative. In certain embodiments, the linker subunit L³,if present, is attached to the camptothecine or camptothecinederivative. In certain embodiments, the linker subunit L⁴, if present,is attached to the camptothecine or camptothecine derivative. In certainembodiments, the linker subunit L⁵, if present, is attached to thecamptothecine or camptothecine derivative. In certain embodiments, thelinker subunit L⁶, if present, is attached to the camptothecine orcamptothecine derivative.

Any convenient linker subunits may be utilized in the linker L. Linkersubunits of interest include, but are not limited to, units of polymerssuch as polyethylene glycols, polyethylenes and polyacrylates, aminoacid residue(s), carbohydrate-based polymers or carbohydrate residuesand derivatives thereof, polynucleotides, alkyl groups, aryl groups,heterocyclic groups, combinations thereof, and substituted versionsthereof. In some embodiments, each of L¹, L², L³, L⁴, L⁵ and L⁶ (ifpresent) comprise one or more groups independently selected from apolyethylene glycol, a modified polyethylene glycol, an amino acidresidue, an alkyl group, a substituted alkyl, an aryl group, asubstituted aryl group, and a diamine (e.g., a linking group thatincludes an alkylene diamine).

In some embodiments, L^(t)(if present) comprises a polyethylene glycol,a modified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, or adiamine. In some embodiments, L^(t) comprises a polyethylene glycol. Insome embodiments, L^(t) comprises a modified polyethylene glycol. Insome embodiments, L¹ comprises an amino acid residue. In someembodiments, L¹ comprises an alkyl group or a substituted alkyl. In someembodiments, L^(t) comprises an aryl group or a substituted aryl group.In some embodiments, L¹ comprises a diamine (e.g., a linking groupcomprising an alkylene diamine).

In some embodiments, L² (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, or adiamine. In some embodiments, L² comprises a polyethylene glycol. Insome embodiments, L² comprises a modified polyethylene glycol. In someembodiments, L² comprises an amino acid residue. In some embodiments, L²comprises an alkyl group or a substituted alkyl. In some embodiments, L²comprises an aryl group or a substituted aryl group. In someembodiments, L² comprises a diamine (e.g., a linking group comprising analkylene diamine).

In some embodiments, L³ (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, or adiamine. In some embodiments, L³ comprises a polyethylene glycol. Insome embodiments, L³ comprises a modified polyethylene glycol. In someembodiments, L³ comprises an amino acid residue. In some embodiments, L³comprises an alkyl group or a substituted alkyl. In some embodiments, L³comprises an aryl group or a substituted aryl group. In someembodiments, L³ comprises a diamine (e.g., a linking group comprising analkylene diamine).

In some embodiments, L⁴ (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, or adiamine. In some embodiments, L⁴ comprises a polyethylene glycol. Insome embodiments, L⁴ comprises a modified polyethylene glycol. In someembodiments, L⁴ comprises an amino acid residue. In some embodiments, L⁴comprises an alkyl group or a substituted alkyl. In some embodiments, L⁴comprises an aryl group or a substituted aryl group. In someembodiments, L⁴ comprises a diamine (e.g., a linking group comprising analkylene diamine).

In some embodiments, L⁵ (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, or adiamine. In some embodiments, L⁵ comprises a polyethylene glycol. Insome embodiments, L⁵ comprises a modified polyethylene glycol. In someembodiments, L⁵ comprises an amino acid residue. In some embodiments, L⁵comprises an alkyl group or a substituted alkyl. In some embodiments, L⁵comprises an aryl group or a substituted aryl group. In someembodiments, L⁵ comprises a diamine (e.g., a linking group comprising analkylene diamine).

In some embodiments, L⁶ (if present) comprises a polyethylene glycol, amodified polyethylene glycol, an amino acid residue, an alkyl group, asubstituted alkyl, an aryl group, a substituted aryl group, or adiamine. In some embodiments, L⁶ comprises a polyethylene glycol. Insome embodiments, L⁶ comprises a modified polyethylene glycol. In someembodiments, L⁶ comprises an amino acid residue. In some embodiments, L⁶comprises an alkyl group or a substituted alkyl. In some embodiments, L⁶comprises an aryl group or a substituted aryl group. In someembodiments, L⁶ comprises a diamine (e.g., a linking group comprising analkylene diamine).

In some embodiments, L is a linker comprising-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-(L⁶)_(f)-, where:

-(L¹)_(a)- is -(T¹-V¹)_(a)-;

-(L²)_(b)- is -(T²-V²)_(b)-;

-(L³)_(c)- is -(T³-V³)_(c)-;

-(L⁴)_(d)- is -(T⁴-V⁴)_(d)-;

-(L⁵)_(e) is -(T⁵-V⁵)_(e)-; and

-(L⁶)_(f)- is -(T⁶-V⁶)_(f)-,

wherein T¹, T², T³, T⁴, T⁵ and T⁶, if present, are tether groups;

V¹, V², V³, V⁴, V⁵ and V⁶, if present, are covalent bonds or linkingfunctional groups; and

a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a,b, c, d, e and f is 1 to 6.

As described above, in certain embodiments, L¹ is attached to thehydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl conjugationmoiety (e.g., as shown in formula (I) above). As such, in certainembodiments, T¹ is attached to the hydrazinyl-indolyl or thehydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown informula (I) above). In certain embodiments, V¹ is attached to thecamptothecine or camptothecine derivative. In certain embodiments, L²,if present, is attached to the camptothecine or camptothecinederivative. As such, in certain embodiments, T², if present, is attachedto the camptothecine or camptothecine derivative, or V², if present, isattached to the camptothecine or camptothecine derivative. In certainembodiments, L³, if present, is attached to the camptothecine orcamptothecine derivative. As such, in certain embodiments, T³, ifpresent, is attached to the camptothecine or camptothecine derivative,or V³, if present, is attached to the camptothecine or camptothecinederivative. In certain embodiments, L⁴, if present, is attached to thecamptothecine or camptothecine derivative. As such, in certainembodiments, T⁴, if present, is attached to the camptothecine orcamptothecine derivative, or V⁴, if present, is attached to thecamptothecine or camptothecine derivative. In certain embodiments, L⁵,if present, is attached to the camptothecine or camptothecinederivative. As such, in certain embodiments, T⁵, if present, is attachedto the camptothecine or camptothecine derivative, or V⁵, if present, isattached to the camptothecine or camptothecine derivative. In certainembodiments, L⁶, if present, is attached to the camptothecine orcamptothecine derivative. As such, in certain embodiments, T⁶, ifpresent, is attached to the camptothecine or camptothecine derivative,or V⁶, if present, is attached to the camptothecine or camptothecinederivative.

Regarding the tether groups, T¹, T², T³, T⁴, T⁵ and T⁶, any convenienttether groups may be utilized in the subject linkers. In someembodiments, T¹, T², T³, T⁴, T⁵ and T⁶ each comprise one or more groupsindependently selected from a covalent bond, a (C₁-C₁₂)alkyl, asubstituted (C₁-C₁₂)alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine (4AP), meta-amino-benzyloxy(MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy(PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB),para-amino-benzylamino (PABA), para-amino-phenyl (PAP),para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide,and an ester, where each w is an integer from 1 to 20, each n is aninteger from 1 to 30, each p is an integer from 1 to 20, and each m isan integer from 1 to 12.

In certain embodiments, the tether group (e.g., T¹, T², T³, T⁴, T⁵and/or T⁶) includes a (C₁-C₁₂)alkyl or a substituted (C₁-C₁₂)alkyl. Incertain embodiments, (C₁-C₁₂)alkyl is a straight chain or branched alkylgroup that includes from 1 to 12 carbon atoms, such as 1 to 10 carbonatoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbonatoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In someinstances, (C₁-C₁₂)alkyl may be an alkyl or substituted alkyl, such asC₁-C₁₂ alkyl, or C₁-C₁₀ alkyl, or C₁-C₆ alkyl, or C₁-C₃ alkyl. In someinstances, (C₁-C₁₂)alkyl is a C₂-alkyl. For example, (C₁-C₁₂)alkyl maybe an alkylene or substituted alkylene, such as C₁-C₁₂ alkylene, orC₁-C₁₀ alkylene, or C₁-C₆ alkylene, or C₁-C₃ alkylene. In someinstances, (C₁-C₁₂)alkyl is a C₂-alkylene (e.g., CH₂CH₂). In someinstances, (C₁-C₁₂)alkyl is a C₃-alkylene (e.g., CH₂CH₂CH₂).

In certain embodiments, substituted (C₁-C₁₂)alkyl is a straight chain orbranched substituted alkyl group that includes from 1 to 12 carbonatoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3carbon atoms. In some instances, substituted (C₁-C₁₂)alkyl may be asubstituted alkyl, such as substituted C₁-C₁₂ alkyl, or substitutedC₁-C₁₀ alkyl, or substituted C₁-C₆ alkyl, or substituted C₁-C₃ alkyl. Insome instances, substituted (C₁-C₁₂)alkyl is a substituted C₂-alkyl. Forexample, substituted (C₁-C₁2)alkyl may be a substituted alkylene, suchas substituted C₁-C₁₂ alkylene, or substituted C₁-C₁₀ alkylene, orsubstituted C₁-C₆ alkylene, or substituted C₁-C₃ alkylene. In someinstances, substituted (C₁-C₁₂)alkyl is a substituted C₂-alkylene. Insome instances, substituted (C₁-C₁2)alkyl is a substituted C₃-alkylene.For example, substituted (C₁-C₁2)alkyl may include C₁-C₁₂ alkylene(e.g., C₃-alkylene or C₅-alkylene) substituted with a (PEG)_(n) group asdescribed herein (e.g., —CONH(PEG)₃ or —NHCO(PEG)₇), or may includeC₁-C₁₂ alkylene (e.g., C₃-alkylene) substituted with a —CONHCH₂CH₂SO₃Hgroup, or may include C₁-C₁₂ alkylene (e.g., C₅-alkylene) substitutedwith a —NHCOCH₂SO₃H group.

In certain embodiments, the tether group (e.g., T¹, T², T³, T⁴, T⁵and/or T⁶) includes an aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, orsubstituted heterocyclyl. In some instances, the tether group (e.g., T¹,T², T³, T⁴, T⁵ and T⁶) includes an aryl or substituted aryl. Forexample, the aryl can be phenyl. In some cases, the substituted aryl isa substituted phenyl. The substituted phenyl can be substituted with oneor more substituents selected from (C₁-C₁₂)alkyl, a substituted(C₁-C₁₂)alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In some instances, the substituted aryl is asubstituted phenyl, where the substituent includes a cleavable moiety asdescribed herein (e.g., an enzymatically cleavable moiety, such as aglycoside or glycoside derivative).

In some instances, the tether group (e.g., T¹, T², T³, T⁴, T⁵ and/or T⁶)includes a heteroaryl or substituted heteroaryl. In some instances, thetether group (e.g., T¹, T², T³, T⁴, T⁵ and T⁶) includes a cycloalkyl orsubstituted cycloalkyl. In some instances, the tether group (e.g., T¹,T², T³, T⁴, T⁵ and T⁶) includes a heterocyclyl or substitutedheterocyclyl. In some instances, the substituent on the substitutedheteroaryl, substituted cycloalkyl or substituted heterocyclyl includesa cleavable moiety as described herein (e.g., an enzymatically cleavablemoiety, such as a glycoside or glycoside derivative).

In certain embodiments, the tether group (e.g., T¹, T², T³, T⁴, T⁵and/or T⁶) includes an ethylene diamine (EDA) moiety, e.g., an EDAcontaining tether group. In certain embodiments, (EDA)_(w) includes oneor more EDA moieties, such as where w is an integer from 1 to 50, suchas from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to6, such as 1, 2, 3, 4, 5 or 6). The linked ethylene diamine (EDA)moieties may optionally be substituted at one or more convenientpositions with any convenient substituents, e.g., with an alkyl, asubstituted alkyl, an acyl, a substituted acyl, an aryl or a substitutedaryl. In certain embodiments, the EDA moiety is described by thestructure:

where y is an integer from 1 to 6, or is 0 or 1, and each R¹² isindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is1, 2, 3, 4, 5 or 6. In certain embodiments, y is 1 and r is 0. Incertain embodiments, y is 1 and r is 1. In certain embodiments, y is 2and r is 0. In certain embodiments, y is 2 and r is 1. In certainembodiments, each R¹² is independently selected from hydrogen, an alkyl,a substituted alkyl, an aryl and a substituted aryl. In certainembodiments, any two adjacent R¹² groups of the EDA may be cyclicallylinked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1and the two adjacent R¹² groups are an alkyl group, cyclically linked toform a piperazinyl ring. In certain embodiments, y is 1 and the adjacentR¹² groups are selected from hydrogen, an alkyl (e.g., methyl) and asubstituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

In certain embodiments, the tether group (e.g., T¹, T², T³, T⁴, T⁵and/or T⁶) includes a 4-amino-piperidine (4AP) moiety (also referred toherein as piperidin-4-amino, P4A). The 4AP moiety may optionally besubstituted at one or more convenient positions with any convenientsubstituents, e.g., with an alkyl, a substituted alkyl, a polyethyleneglycol moiety, an acyl, a substituted acyl, an aryl or a substitutedaryl. In certain embodiments, the 4AP moiety is described by thestructure:

where R¹² is selected from hydrogen, alkyl, substituted alkyl, apolyethylene glycol moiety (e.g., a polyethylene glycol or a modifiedpolyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R¹² is a polyethylene glycol moiety. In certainembodiments, R¹² is a carboxy modified polyethylene glycol.

In certain embodiments, R¹² includes a polyethylene glycol moietydescribed by the formula: (PEG)_(k), which may be represented by thestructure:

where k is an integer from 1 to 20, such as from 1 to 18, or from 1 to16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8,or from 1 to 6, or from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, kis 2. In certain embodiments, R¹⁷ is selected from OH, OR, COOH, orCOOR, where R is selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl. In certainembodiments, R¹⁷ is COOH. In certain embodiments, R¹⁷ is OH. In certainembodiments, R¹⁷ is OR, such as OCH₃.

In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴, T⁵ and/orT⁶) includes (PEG)_(n), where (PEG)_(n) is a polyethylene glycol or amodified polyethylene glycol linking unit. In certain embodiments,(PEG)_(n) is described by the structure:

where n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30,from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, nis 2. In some instances, n is 3. In some instances, n is 6. In someinstances, n is 12.

In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴, T⁵ and/orT⁶) includes (AA)_(p), where AA is an amino acid residue. Any convenientamino acids may be utilized. Amino acids of interest include but are notlimited to, L- and D-amino acids, naturally occurring amino acids suchas any of the 20 primary alpha-amino acids and beta-alanine,non-naturally occurring amino acids (e.g., amino acid analogs), such asa non-naturally occurring alpha-amino acid or a non-naturally occurringbeta-amino acid, etc. In certain embodiments, p is an integer from 1 to50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 orfrom 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certainembodiments, p is 2.

In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴, T⁵ and/orT⁶) includes an amino acid analog. Amino acid analogs include compoundsthat are similar in structure and/or overall shape to one or more aminoacids commonly found in naturally occurring proteins (e.g., Ala or A,Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I,Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R,Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogsalso include natural amino acids with modified side chains or backbones.Amino acid analogs also include amino acid analogs with the samestereochemistry as in the naturally occurring D-form, as well as theL-form of amino acid analogs. In some instances, the amino acid analogsshare backbone structures, and/or the side chain structures of one ormore natural amino acids, with difference(s) being one or more modifiedgroups in the molecule. Such modification may include, but is notlimited to, substitution of an atom (such as N) for a related atom (suchas S), addition of a group (such as methyl, or hydroxyl, etc.) or anatom (such as Cl or Br, etc.), deletion of a group, substitution of acovalent bond (single bond for double bond, etc.), or combinationsthereof. For example, amino acid analogs may include α-hydroxy acids,and α-amino acids, and the like. Examples of amino acid analogs include,but are not limited to, sulfoalanine, and the like.

In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴, T⁵ and/orT⁶) includes a moiety described by the formula —(CR¹³OH)_(m)—, where mis 0 or n is an integer from 1 to 50, such as from 1 to 40, from 1 to30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12. In certain embodiments, m is 1. In certainembodiments, m is 2. In certain embodiments, R¹³ is selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R¹³ is hydrogen. Incertain embodiments, R¹³ is alkyl or substituted alkyl, such as C₁₋₆alkyl or C₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substitutedalkyl, or C₁₋₃ alkyl or C₁₋₃ substituted alkyl. In certain embodiments,R¹³ is alkenyl or substituted alkenyl, such as C₂₋₆ alkenyl or C₂₋₆substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, orC₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certain embodiments, R¹³ isalkynyl or substituted alkynyl. In certain embodiments, R¹³ is alkoxy orsubstituted alkoxy. In certain embodiments, R¹³ is amino or substitutedamino. In certain embodiments, R¹³ is carboxyl or carboxyl ester. Incertain embodiments, R¹³ is acyl or acyloxy. In certain embodiments, R¹³is acyl amino or amino acyl. In certain embodiments, R¹³ is alkylamideor substituted alkylamide. In certain embodiments, R¹³ is sulfonyl. Incertain embodiments, R¹³ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R¹³ is aryl or substituted aryl, such as C₅₋₈ arylor C₅₋₈ substituted aryl, such as a C₅ aryl or C₅ substituted aryl, or aC₆ aryl or C₆ substituted aryl. In certain embodiments, R¹³ isheteroaryl or substituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈substituted heteroaryl, such as a C₅ heteroaryl or C₅ substitutedheteroaryl, or a C₆ heteroaryl or C₆ substituted heteroaryl. In certainembodiments, R¹³ is cycloalkyl or substituted cycloalkyl, such as C₃₋₈cycloalkyl or C₃₋₈ substituted cycloalkyl, such as a C₃₋₆ cycloalkyl orC₃₋₆ substituted cycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅ substitutedcycloalkyl. In certain embodiments, R¹³ is heterocyclyl or substitutedheterocyclyl, such as C₃₋₈ heterocyclyl or C₃₋₈ substitutedheterocyclyl, such as a C₃₋₆ heterocyclyl or C₃₋₆ substitutedheterocyclyl, or a C₃₋₅ heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R¹³ is selected from hydrogen, alkyl,substituted alkyl, aryl, and substituted aryl. In these embodiments,alkyl, substituted alkyl, aryl, and substituted aryl are as describedabove for R¹³.

In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴, T⁵ and/orT⁶) includes a meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl(MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl(PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA),para-amino-phenyl (PAP), or para-hydroxy-phenyl (PHP).

In some embodiments, a tether includes a MABO group described by thefollowing structure:

In some embodiments, a tether includes a MABC group described by thefollowing structure:

In some embodiments, a tether includes a PABO group described by thefollowing structure:

In some embodiments, a tether includes a PABC group described by thefollowing structure:

In some embodiments, a tether includes a PAB group described by thefollowing structure:

In some embodiments, a tether includes a PABA group described by thefollowing structure:

In some embodiments, a tether includes a PAP group described by thefollowing structure:

In some embodiments, a tether includes a PHP group described by thefollowing structure:

In certain embodiments, each R¹⁴ is independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.

In certain embodiments, R¹⁴ is hydrogen. In certain embodiments, eachR¹⁴ is hydrogen. In certain embodiments, R¹⁴ is alkyl or substitutedalkyl, such as C₁₋₆ alkyl or C₁₋₆ substituted alkyl, or C₁₋₄ alkyl orC₁₋₄ substituted alkyl, or C₁₋₃ alkyl or C₁₋₃ substituted alkyl. Incertain embodiments, R¹⁴ is alkenyl or substituted alkenyl, such as C₂₋₆alkenyl or C₂₋₆ substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substitutedalkenyl, or C₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certainembodiments, R¹⁴ is alkynyl or substituted alkynyl. In certainembodiments, R¹⁴ is alkoxy or substituted alkoxy. In certainembodiments, R¹⁴ is amino or substituted amino. In certain embodiments,R¹⁴ is carboxyl or carboxyl ester. In certain embodiments, R¹⁴ is acylor acyloxy. In certain embodiments, R¹⁴ is acyl amino or amino acyl. Incertain embodiments, R¹⁴ is alkylamide or substituted alkylamide. Incertain embodiments, R¹⁴ is sulfonyl. In certain embodiments, R¹⁴ isthioalkoxy or substituted thioalkoxy. In certain embodiments, R¹⁴ isaryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl,such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substitutedaryl. In certain embodiments, R¹⁴ is heteroaryl or substitutedheteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, suchas a C₅ heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl orC₆ substituted heteroaryl. In certain embodiments, R¹⁴ is cycloalkyl orsubstituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R¹⁴ is heterocyclyl or substituted heterocyclyl, such asC₃₋₈ heterocyclyl or C₃₋₈ substituted heterocyclyl, such as a C₃₋₆heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅ heterocyclyl orC₃₋₅ substituted heterocyclyl.

In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, andPHP tether structures shown above, the phenyl ring may be substitutedwith one or more additional groups selected from halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments of the linker L, one or more of the tether groupsT¹, T², T³, T⁴, T⁵ or T⁶ is each optionally substituted with a glycosideor glycoside derivative. In certain embodiments, the glycoside orglycoside derivative is selected from a glucuronide, a galactoside, aglucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

In certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, andPHP tether structures shown above may be substituted with an one or moreadditional groups selected from a glycoside and a glycoside derivative.For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB,PABA, PAP, and PHP tether structures shown above, the phenyl ring may besubstituted with one or more additional groups selected from a glycosideand a glycoside derivative. In certain embodiments, the glycoside orglycoside derivative is selected from a glucuronide, a galactoside, aglucoside, a mannoside, a fucoside, O-GlcNAc, and 0-GalNAc.

For example, in some embodiments, the glycoside or glycoside derivativecan be selected from the following structures:

Regarding the linking functional groups, V¹, V², V³, V⁴, V⁵ and V⁶, anyconvenient linking functional groups may be utilized in the linker L.Linking functional groups of interest include, but are not limited to,amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide,sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate,thiophosphoraidate, and the like. In some embodiments, V¹, V², V³, V⁴,V⁵ and V⁶ are each independently selected from a covalent bond, —CO—,—NR¹⁵—, —NR¹⁵(CH₂)_(q)—, —NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—,—OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and —P(O)OH—,where q is an integer from 1 to 6. In certain embodiments, q is aninteger from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6). In certain embodiments,q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3.In certain embodiments, q is 4. In certain embodiments, q is 5. Incertain embodiments, q is 6.

In some embodiments, each R¹⁵ is independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl.

In certain embodiments, R¹⁵ is hydrogen. In certain embodiments, eachR¹⁵ is hydrogen. In certain embodiments, R¹⁵ is alkyl or substitutedalkyl, such as C₁₋₆ alkyl or C₁₋₆ substituted alkyl, or C₁₋₄ alkyl orC₁₋₄ substituted alkyl, or C₁₋₃ alkyl or C₁₋₃ substituted alkyl. Incertain embodiments, R¹⁵ is alkenyl or substituted alkenyl, such as C₂₋₆alkenyl or C₂₋₆ substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substitutedalkenyl, or C₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certainembodiments, R¹⁵ is alkynyl or substituted alkynyl. In certainembodiments, R¹⁵ is alkoxy or substituted alkoxy. In certainembodiments, R¹⁵ is amino or substituted amino. In certain embodiments,R¹⁵ is carboxyl or carboxyl ester. In certain embodiments, R¹⁵ is acylor acyloxy. In certain embodiments, R¹⁵ is acyl amino or amino acyl. Incertain embodiments, R¹⁵ is alkylamide or substituted alkylamide. Incertain embodiments, R¹⁵ is sulfonyl. In certain embodiments, R¹⁵ isthioalkoxy or substituted thioalkoxy. In certain embodiments, R¹⁵ isaryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl,such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substitutedaryl. In certain embodiments, R¹⁵ is heteroaryl or substitutedheteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, suchas a C₅ heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl orC₆ substituted heteroaryl. In certain embodiments, R¹⁵ is cycloalkyl orsubstituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R¹⁵ is heterocyclyl or substituted heterocyclyl, such asC₃₋₈ heterocyclyl or C₃₋₈ substituted heterocyclyl, such as a C₃₋₆heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅ heterocyclyl orC₃₋₅ substituted heterocyclyl.

In certain embodiments, each R¹⁵ is independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Inthese embodiments, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl areas described above for R¹⁵.

In certain embodiments, the tether group includes an acetal group, adisulfide, a hydrazine, or an ester. In some embodiments, the tethergroup includes an acetal group. In some embodiments, the tether groupincludes a hydrazine. In some embodiments, the tether group includes adisulfide. In some embodiments, the tether group includes an ester.

As described above, in some embodiments, L is a linker comprising-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)-(T⁶-V⁶)_(f)-,where a, b, c, d, e and f are each independently 0 or 1, where the sumof a, b, c, d, e and f is 1 to 6.

In some embodiments, in the linker L:

T¹ is selected from a (C₁-C₁₂)alkyl and a substituted (C₁-C₁₂)alkyl;

T², T³, T⁴, T⁵ and T⁶ are each independently selected from(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine (4AP), MABO, MABC, PABO,PABC, PAB, PABA, PAP, PHP, an acetal group, a disulfide, a hydrazine,and an ester; and

V¹, V², V³, V⁴, V⁵ and V⁶ are each independently selected from acovalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—, —NR¹⁵(C₆H₄)—, —CONR¹⁵—,—NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂-, —SO₂NR¹⁵—,—NR¹⁵SO₂— and —P(O)OH—, wherein q is an integer from 1 to 6;

wherein:

(PEG)_(n) is

where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4AP) is

AA is an amino acid residue, where p is an integer from 1 to 20; and

each R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a polyethylene glycol moiety, an aryl and asubstituted aryl, wherein any two adjacent R¹² groups may be cyclicallylinked to form a piperazinyl ring;

each R¹³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, and substituted aryl; and

each R¹⁵ is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In certain embodiments, T¹, T², T³, T⁴, T⁵ and T⁶ and V¹, V², V³, V⁴, V⁵and V⁶ are selected from the following:

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

T⁶ is EDA and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is absent and V⁵ is —NR¹⁵(C₆H₄)—; and

T⁶ is absent and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is —NR¹⁵—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent;

T⁴ is EDA and V⁴ is —CO—; and

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent; and

d, e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABA and V⁵ is —CO—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —SO₂—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is substituted (C₁-C₁₂)alkyl and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

T⁵ is (C₁-C₁₂)alkyl and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —O—;

T⁴ is (C₁-C₁₂)alkyl and V⁴ is —CO—;

T⁵ is AA and V⁵ is absent;

T⁶ is PABC and V⁶ is absent; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is absent;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CONH—;

T³ is substituted (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABO and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PAP and V⁵ is —COO—; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PAP and V⁴ is —COO—; and

e and f are each 0.

In certain embodiments, the left-hand side of the linker structure isattached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinylconjugation moiety, and the right-hand side of the linker structure isattached to the camptothecine or a camptothecine derivative.

In certain embodiments of the conjugate of formula (I), the linker L isattached to a camptothecine or a camptothecine derivative. In someinstances, the linker L is attached to a compound of formula (II) at R¹,R², R³, R⁴, R⁵ or R⁶:

wherein:

R¹ and R² are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R¹ and R² are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R³ and R⁴ are each independently selected from hydrogen, halo, hydroxy,amino, substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR³ and R⁴ are optionally cyclically linked to form a 5 or 6-memberedcycloalkyl or heterocyclyl ring;

R⁵ is selected from hydrogen, halogen, hydroxy, amino, substitutedamino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

R⁶ is selected from OH and OC(O)R¹¹; and

R¹¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl, wherein at leastone R¹⁰ is optionally linked to a second compound of formula (II).

In certain embodiments, R¹ and R² are each independently selected fromhydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or R¹ and R² are optionallycyclically linked to form a 5 or 6-membered cycloalkyl or heterocyclylring.

In certain embodiments, R¹ is selected from hydrogen, halogen, hydroxy,amino, substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R¹ is hydrogen. In certain embodiments, R¹ ishalogen (e.g., F, Cl, Br, I). In certain embodiments, R¹ is hydroxy. Incertain embodiments, R¹ is amino or substituted amino. In certainembodiments, R¹ is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R¹ is methyl.In certain embodiments, R¹ is alkenyl or substituted alkenyl, such asC₂₋₆ alkenyl or C₂₋₆ substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄substituted alkenyl, or C₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. Incertain embodiments, R¹ is alkynyl or substituted alkynyl. In certainembodiments, R¹ is alkoxy or substituted alkoxy. In certain embodiments,R¹ is aryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substitutedaryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆substituted aryl. In certain embodiments, R¹ is heteroaryl orsubstituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substitutedheteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or aC₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R¹is cycloalkyl or substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈substituted cycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substitutedcycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. Incertain embodiments, R¹ is heterocyclyl or substituted heterocyclyl,such as a C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R² is selected from hydrogen, halogen, hydroxy,amino, substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R² is hydrogen. In certain embodiments, R² ishalogen (e.g., F, Cl, Br, I). In certain embodiments, R² is hydroxy. Incertain embodiments, R² is amino or substituted amino. In certainembodiments, R² is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R² is methyl.In certain embodiments, R² is alkenyl or substituted alkenyl, such asC₂₋₆ alkenyl or C₂₋₆ substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄substituted alkenyl, or C₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. Incertain embodiments, R² is alkynyl or substituted alkynyl. In certainembodiments, R² is alkoxy or substituted alkoxy. In certain embodiments,R² is aryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substitutedaryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆substituted aryl. In certain embodiments, R² is heteroaryl orsubstituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substitutedheteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or aC₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R²is cycloalkyl or substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈substituted cycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substitutedcycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. Incertain embodiments, R² is heterocyclyl or substituted heterocyclyl,such as a C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R¹ and R² are optionally cyclically linked toform a 5 or 6-membered cycloalkyl or heterocyclyl ring. In certainembodiments, R¹ and R² are cyclically linked to form a 5 or 6-memberedcycloalkyl. In certain embodiments, R¹ and R² are cyclically linked toform a 5 or 6-membered heterocyclyl. In certain embodiments, R¹ and R²are cyclically linked to form a 5-membered cycloalkyl. In certainembodiments, R¹ and R² are cyclically linked to form a 6-memberedcycloalkyl. In certain embodiments, R¹ and R² are cyclically linked toform a 5-membered heterocyclyl. In certain embodiments, R¹ and R² arecyclically linked to form a 6-membered heterocyclyl.

In certain embodiments, R³ and R⁴ are each independently selected fromhydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or R³ and R⁴ are optionallycyclically linked to form a 5 or 6-membered cycloalkyl or heterocyclylring.

In certain embodiments, R³ is selected from hydrogen, halogen, hydroxy,amino, substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R³ is hydrogen. In certain embodiments, R³ ishalogen (e.g., F, Cl, Br, I). In certain embodiments, R³ is hydroxy. Incertain embodiments, R³ is amino or substituted amino. In certainembodiments, R³ is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R³ is methyl.In certain embodiments, R³ is alkenyl or substituted alkenyl, such asC₂₋₆ alkenyl or C₂₋₆ substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄substituted alkenyl, or C₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. Incertain embodiments, R³ is alkynyl or substituted alkynyl. In certainembodiments, R³ is alkoxy or substituted alkoxy. In certain embodiments,R³ is aryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substitutedaryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆substituted aryl. In certain embodiments, R³ is heteroaryl orsubstituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substitutedheteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or aC₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R³is cycloalkyl or substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈substituted cycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substitutedcycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. Incertain embodiments, R³ is heterocyclyl or substituted heterocyclyl,such as a C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R⁴ is selected from hydrogen, halogen, hydroxy,amino, substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁴ is hydrogen. In certain embodiments, R⁴ ishalogen (e.g., F, Cl, Br, I). In certain embodiments, R⁴ is hydroxy. Incertain embodiments, R⁴ is amino or substituted amino. In certainembodiments, R⁴ is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R⁴ is methyl.In certain embodiments, R⁴ is alkenyl or substituted alkenyl, such asC₂₋₆ alkenyl or C₂₋₆ substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄substituted alkenyl, or C₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. Incertain embodiments, R⁴ is alkynyl or substituted alkynyl. In certainembodiments, R⁴ is alkoxy or substituted alkoxy. In certain embodiments,R⁴ is aryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substitutedaryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆substituted aryl. In certain embodiments, R⁴ is heteroaryl orsubstituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substitutedheteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or aC₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R⁴is cycloalkyl or substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈substituted cycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substitutedcycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₈ substituted cycloalkyl. Incertain embodiments, R⁴ is heterocyclyl or substituted heterocyclyl,such as a C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R³ and R⁴ are optionally cyclically linked toform a 5 or 6-membered cycloalkyl or heterocyclyl ring. In certainembodiments, R³ and R⁴ are cyclically linked to form a 5 or 6-memberedcycloalkyl. In certain embodiments, R³ and R⁴ are cyclically linked toform a 5 or 6-membered heterocyclyl. In certain embodiments, R³ and R⁴are cyclically linked to form a 5-membered cycloalkyl. In certainembodiments, R³ and R⁴ are cyclically linked to form a 6-memberedcycloalkyl. In certain embodiments, R³ and R⁴ are cyclically linked toform a 5-membered heterocyclyl. In certain embodiments, R³ and R⁴ arecyclically linked to form a 6-membered heterocyclyl.

In certain embodiments, R⁵ is selected from hydrogen, halogen, hydroxy,amino, substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁵ is hydrogen. In certain embodiments, R⁵ ishalogen (e.g., F, Cl, Br, I). In certain embodiments, R⁵ is hydroxy. Incertain embodiments, R⁵ is amino or substituted amino. In certainembodiments, R⁵ is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R⁵ is methyl.In certain embodiments, R⁵ is alkenyl or substituted alkenyl, such asC₂₋₆ alkenyl or C₂₋₆ substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄substituted alkenyl, or C₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. Incertain embodiments, R⁵ is alkynyl or substituted alkynyl. In certainembodiments, R⁵ is alkoxy or substituted alkoxy. In certain embodiments,R⁵ is aryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substitutedaryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆substituted aryl. In certain embodiments, R⁵ is heteroaryl orsubstituted heteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substitutedheteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or aC₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R⁵is cycloalkyl or substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈substituted cycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substitutedcycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. Incertain embodiments, R⁵ is heterocyclyl or substituted heterocyclyl,such as a C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl.

In certain embodiments, R⁶ is selected from OH and OC(O)R¹¹. In certainembodiments, R⁶ is OH. In certain embodiments, R⁶ is OC(O)R¹¹.

In certain embodiments, R¹¹ is selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, R¹¹ is hydrogen. In certainembodiments, R¹¹ is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R¹¹ is alkenylor substituted alkenyl, such as C₂₋₆ alkenyl or C₂₋₆ substitutedalkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, or C₂₋₃ alkenyl orC₂₋₃ substituted alkenyl. In certain embodiments, R¹¹ is alkynyl orsubstituted alkynyl. In certain embodiments, R¹¹ is aryl or substitutedaryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl, such as a C₅ aryl orC₅ substituted aryl, or a C₆ aryl or C₆ substituted aryl. In certainembodiments, R¹¹ is heteroaryl or substituted heteroaryl, such as C₅₋₈heteroaryl or C₅₋₈ substituted heteroaryl, such as a C₅ heteroaryl or C₅substituted heteroaryl, or a C₆ heteroaryl or C₆ substituted heteroaryl.In certain embodiments, R¹¹ is cycloalkyl or substituted cycloalkyl,such as C₃₋₈ cycloalkyl or C₃₋₈ substituted cycloalkyl, such as a C₃₋₆cycloalkyl or C₃₋₆ substituted cycloalkyl, or a C₃₋₅ cycloalkyl or C₃₋₅substituted cycloalkyl. In certain embodiments, R¹¹ is heterocyclyl orsubstituted heterocyclyl, such as a C₃₋₆ heterocyclyl or C₃₋₆substituted heterocyclyl, or a C₃₋₅ heterocyclyl or C₃₋₅ substitutedheterocyclyl.

In certain embodiments, the compound of formula (II) has the structureof formula (IIa):

In certain embodiments of the compound of formula (IIa), R³ is asdescribed above.

In certain embodiments of the compound of formula (IIa), R⁶ is asdescribed above.

In certain embodiments of the compound of formula (IIa), R³ is OH and Lis attached at R⁶. In certain embodiments of the compound of formula(IIa), L is attached at R³ and R⁶ is OH.

In certain embodiments, the compound of formula (II) has the structureof formula (IIb):

In certain embodiments of the compound of formula (IIb), R^(1a) isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl,and sulfonyl. In certain embodiments, R^(1a) is hydrogen. In certainembodiments, R^(1a) is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R^(1a) is arylor substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl, such asa C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substituted aryl.In certain embodiments, R^(1a) is heteroaryl or substituted heteroaryl,such as C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, such as a C₅heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆substituted heteroaryl. In certain embodiments, R^(1a) is cycloalkyl orsubstituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R^(1a) is heterocyclyl or substituted heterocyclyl, such asa C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl. In certain embodiments,R^(1a) is carboxyl. In certain embodiments, R^(1a) is carboxyl ester. Incertain embodiments, R^(1a) is acyl. In certain embodiments, R^(1a) issulfonyl.

In certain embodiments of the compound of formula (IIb), R⁶ is asdescribed above.

In certain embodiments of the compound of formula (IIb), R^(1a) isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl,and sulfonyl, and L is attached at R⁶. In certain embodiments of thecompound of formula (IIb), L is attached at R^(1a) and R⁶ is OH.

In certain embodiments, the compound of formula (II) has the structureof formula (IIc):

In certain embodiments of the compound of formula (IIc), R^(1b) isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl,and sulfonyl. In certain embodiments, R^(1b) is hydrogen. In certainembodiments, R^(1b) is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R^(1b) is arylor substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl, such asa C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substituted aryl.In certain embodiments, R^(1b) is heteroaryl or substituted heteroaryl,such as C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, such as a C₅heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆substituted heteroaryl. In certain embodiments, R^(1b) is cycloalkyl orsubstituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R^(1b) is heterocyclyl or substituted heterocyclyl, such asa C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl. In certain embodiments,R^(1b) is carboxyl. In certain embodiments, R^(1b) is carboxyl ester. Incertain embodiments, R^(1b) is acyl. In certain embodiments, R^(1b) issulfonyl.

In certain embodiments of the compound of formula (IIc), R⁶ is asdescribed above.

In certain embodiments of the compound of formula (IIc), R^(1b) isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl,and sulfonyl, and L is attached at R⁶. In certain embodiments of thecompound of formula (IIc), L is attached at R^(1b) and R⁶ is OH.

In certain embodiments, the compound of formula (II) has the structureof formula (IId):

In certain embodiments of the compound of formula (IId), R^(2a) andR^(2b) are each independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl.

In certain embodiments of the compound of formula (IId), R^(2a) isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl,and sulfonyl. In certain embodiments, R^(2a) is hydrogen. In certainembodiments, R^(2a) is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R^(2a) is arylor substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl, such asa C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substituted aryl.In certain embodiments, R^(2a) is heteroaryl or substituted heteroaryl,such as C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, such as a C₅heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆substituted heteroaryl. In certain embodiments, R^(2a) is cycloalkyl orsubstituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R^(2a) is heterocyclyl or substituted heterocyclyl, such asa C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl. In certain embodiments,R^(2a) is carboxyl. In certain embodiments, R^(2a) is carboxyl ester. Incertain embodiments, R^(2a) is acyl. In certain embodiments, R^(2a) issulfonyl.

In certain embodiments of the compound of formula (IId), R^(2b) isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl,and sulfonyl. In certain embodiments, R^(2b) is hydrogen. In certainembodiments, R^(2b) is alkyl or substituted alkyl, such as C₁₋₆ alkyl orC₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substituted alkyl, or C₁₋₃alkyl or C₁₋₃ substituted alkyl. In certain embodiments, R^(2b) is arylor substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl, such asa C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substituted aryl.In certain embodiments, R^(2b) is heteroaryl or substituted heteroaryl,such as C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, such as a C₅heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆substituted heteroaryl. In certain embodiments, R^(2b) is cycloalkyl orsubstituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R^(2b) is heterocyclyl or substituted heterocyclyl, such asa C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl. In certain embodiments,R^(2b) is carboxyl. In certain embodiments, R^(2b) is carboxyl ester. Incertain embodiments, R^(2b) is acyl. In certain embodiments, R^(2b) issulfonyl.

In certain embodiments of the compound of formula (IId), R⁶ is asdescribed above.

In certain embodiments of the compound of formula (IId), R^(2a) andR^(2b) are each independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶.In certain embodiments of the compound of formula (IId), L is attachedat R^(2a) or R^(2b) and R⁶ is OH. In certain embodiments of the compoundof formula (IId), L is attached at R^(2a) and R⁶ is OH. In certainembodiments of the compound of formula (IId), L is attached at R^(2b)and R⁶ is OH.

In certain embodiments, the compound of formula (II) has the structureof formula (IIe):

In certain embodiments of the compound of formula (IIe), R^(2c) isselected from alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, carboxyl, carboxyl ester, acyl,and sulfonyl, and attachment to L is indicated by the wavy line.

In certain embodiments, R^(2c) is alkyl or substituted alkyl, such asC₁₋₆ alkyl or C₁₋₆ substituted alkyl, or C₁₋₄ alkyl or C₁₋₄ substitutedalkyl, or C₁₋₃ alkyl or C₁₋₃ substituted alkyl. In certain embodiments,R^(2c) is alkenyl or substituted alkenyl, such as C₂₋₆ alkenyl or C₂₋₆substituted alkenyl, or C₂₋₄ alkenyl or C₂₋₄ substituted alkenyl, orC₂₋₃ alkenyl or C₂₋₃ substituted alkenyl. In certain embodiments, R^(2c)is alkynyl or substituted alkynyl, such as C₂₋₆ alkynyl or C₂₋₆substituted alkynyl, or C₂₋₄ alkynyl or C₂₋₄ substituted alkynyl, orC₂₋₃ alkynyl or C₂₋₃ substituted alkynyl. In certain embodiments, R^(2c)is aryl or substituted aryl, such as C₅₋₈ aryl or C₅₋₈ substituted aryl,such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substitutedaryl. In certain embodiments, R^(2c) is heteroaryl or substitutedheteroaryl, such as C₅₋₈ heteroaryl or C₅₋₈ substituted heteroaryl, suchas a C₅ heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl orC₆ substituted heteroaryl. In certain embodiments, R^(2c) is cycloalkylor substituted cycloalkyl, such as C₃₋₈ cycloalkyl or C₃₋₈ substitutedcycloalkyl, such as a C₃₋₆ cycloalkyl or C₃₋₆ substituted cycloalkyl, ora C₃₋₅ cycloalkyl or C₃₋₅ substituted cycloalkyl. In certainembodiments, R^(2c) is heterocyclyl or substituted heterocyclyl, such asa C₃₋₆ heterocyclyl or C₃₋₆ substituted heterocyclyl, or a C₃₋₅heterocyclyl or C₃₋₅ substituted heterocyclyl. In certain embodiments,R^(2e) is carboxyl. In certain embodiments, R^(2c) is carboxyl ester. Incertain embodiments, R^(2c) is acyl. In certain embodiments, R^(2c) issulfonyl.

In certain embodiments of the compound of formula (IIe), attachment to Lis indicated by the wavy line. Stated another way, the bond with thewavy line indicates the bond that attaches the compound of formula (IIe)to the linker.

In certain embodiments of the conjugate of formula (I), the polypeptide(e.g., antibody) can be linked to one drug or active agent through theconjugation moiety. In some instances, the polypeptide (e.g., antibody)can be linked to more than one drug or active agent through theconjugation moiety. For example, the conjugation moiety can be linked totwo or more drugs or active agents. Each drug or active agent can belinked via a corresponding linker to the same conjugation moiety, whichin turn can be attached to a polypeptide (e.g., antibody) as describedherein, thus linking the polypeptide (e.g., antibody) to two or moredrugs or active agents.

For example, in certain embodiments of the conjugate of formula (I), oneor more R¹⁰ is optionally linked to a second compound of formula (II).In some cases, one or more R¹⁰ is linked to a second compound of formula(II). In other cases, R¹⁰ is not linked to a second compound of formula(II). For example, at least one R¹⁰ is optionally linked to a secondcompound of formula (II). In some instances, one R¹⁰ is linked to asecond compound of formula (II).

In certain embodiments, one R¹⁰ is linked via a second linker, L², to asecond compound of formula (II). In certain embodiments, the secondlinker L² is a linker (e.g., a second linker) described by the formula:

-(L⁷)_(g)-(L⁸)_(h)-(L⁹)_(i)-(L¹⁰)_(j)-(L¹¹)_(k)-(L¹²)₁-,

wherein L⁷, L⁸, L⁹, L¹⁰, L¹¹ and L¹² are each independently a linkersubunit, and g, h, i, j, k and l are each independently 0 or 1, whereinthe sum of g, h, i, j, k and l is 1 to 6.

In certain embodiments, the sum of g, h, i, j, k and l is 1. In certainembodiments, the sum of g, h, i, j, k and l is 2. In certainembodiments, the sum of g, h, i, j, k and l is 3. In certainembodiments, the sum of g, h, i, j, k and l is 4. In certainembodiments, the sum of g, h, i, j, k and l is 5. In certainembodiments, the sum of g, h, i, j, k and l is 6. In certainembodiments, g, h, i, j, k and l are each 1. In certain embodiments, g,h, i, j and k are each 1 and l is 0. In certain embodiments, g, h, i andj are each 1 and k and l are each 0. In certain embodiments, g, h, and iare each 1 and j, k and l are each 0. In certain embodiments, g and hare each 1 and i, j, k and 1 are each 0. In certain embodiments, g is 1and h, i, j, k and l are each 0.

In certain embodiments, the linker subunit L⁷ is attached to thehydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl conjugationmoiety (e.g., as shown in formula (I) above). In certain embodiments,the linker subunit L⁸, if present, is attached to the camptothecine orcamptothecine derivative. In certain embodiments, the linker subunit L⁹,if present, is attached to the camptothecine or camptothecinederivative. In certain embodiments, the linker subunit L¹⁰, if present,is attached to the camptothecine or camptothecine derivative. In certainembodiments, the linker subunit L¹¹, if present, is attached to thecamptothecine or camptothecine derivative. In certain embodiments, thelinker subunit L¹², if present, is attached to the camptothecine orcamptothecine derivative.

Any convenient linker subunits may be utilized in the second linkerL^(B). For example, any of the linker subunits described above inrelation to L¹, L², L³, L⁴, L⁵ and L⁶ may be used for the linkersubunits L⁷, L⁸, L⁹, L¹⁰, L¹¹ and L¹².

In certain embodiments, the second linker L^(B) is a linker comprising-(L⁷)_(g)-(L⁸)_(h)-(L⁹)_(i)-(L¹⁰)_(j)(L¹¹)_(k)-(L¹²)₁-, where:

-(L⁷)_(g)- is -(T⁷-V⁷)_(g)-;

-(L⁸)_(h)- is -(T⁸-V⁸)_(h)-;

-(L⁹)_(i)- is -(T⁹-V⁹)_(i)-;

-(L¹⁰)_(j)- is -(T¹⁰-V¹⁰)_(j)-;

-(L¹¹)_(k)- is -(T¹¹-V¹¹)_(k)-; and

-(L¹²)_(i)- is -(T¹²-V¹²)_(i)-,

wherein T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹², if present, are tether groups;

V⁷, V⁸, V⁹, V¹⁰, V¹¹ and V¹², if present, are covalent bonds or linkingfunctional groups; and

g, h, i, j, k and l are each independently 0 or 1, wherein the sum of g,h, i, j, k and l is 1 to 6.

Accordingly, in certain embodiments, the second linker L^(B) comprises:

-(T⁷-V⁷)_(g)-(T⁸-V⁸)_(h)-(T⁹-V⁹)_(i)-(T¹⁰-V¹⁰)_(j)-(T¹¹-V¹)_(k)-(T¹²-V¹²)—,

wherein

g, h, i, j, k and l are each independently 0 or 1;

T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² are each independently selected from acovalent bond, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl,(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine(4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC),para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC),para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl(PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, adisulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEGis a polyethylene glycol, and AA is an amino acid residue or an aminoacid analog, wherein each w is an integer from 1 to 20, each n is aninteger from 1 to 30, each p is an integer from 1 to 20, and each m isan integer from 1 to 12;

V⁷, V⁸, V⁹, V¹⁰, V¹¹ and V¹² are each independently selected from thegroup consisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH2)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and —P(O)OH—, wherein each q is an integerfrom 1 to 6;

each R¹³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, and substituted aryl; and

each R¹⁵ is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

Any convenient tether groups may be utilized for T⁷, T⁸, T⁹, T¹⁰, T¹¹and T¹². For example, any of the tether groups described above inrelation to T¹, T², T³, T⁴, T⁵ and T⁶ may be used for the tether groupsT⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹².

Any convenient linking functional groups may be utilized for V⁷, V⁸, V⁹,V¹⁰, V¹¹ and V¹². For example, any of the linking functional groupsdescribed above in relation to V¹, V², V³, V⁴, V⁵ and V⁶ may be used forthe linking functional groups V⁷, V⁸, V⁹, V¹⁰, V¹¹ and V¹².

In certain embodiments, each R¹³ is independently selected fromhydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In theseembodiments, alkyl, substituted alkyl, aryl, and substituted aryl are asdescribed above for R¹³.

In certain embodiments, each R¹⁵ is independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Inthese embodiments, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl areas described above for R¹⁵. In these embodiments, various possiblesubstituents are as described above for R¹⁵.

In certain embodiments of the second linker L^(B), one or more of thetether groups T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² is each optionallysubstituted with a glycoside or glycoside derivative. In certainembodiments, the glycoside or glycoside derivative is selected from aglucuronide, a galactoside, a glucoside, a mannoside, a fucoside,O-GlcNAc, and O-GalNAc.

In certain embodiments of the second linker L^(B), the MABO, MABC, PABO,PABC, PAB, PABA, PAP, and PHP tether structures shown above may besubstituted with an one or more additional groups selected from aglycoside and a glycoside derivative. For example, in some embodimentsof the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structuresshown above, the phenyl ring may be substituted with one or moreadditional groups selected from a glycoside and a glycoside derivative.In certain embodiments, the glycoside or glycoside derivative isselected from a glucuronide, a galactoside, a glucoside, a mannoside, afucoside, O-GlcNAc, and O-GalNAc.

In certain embodiments, T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² and V⁷, V⁸, V⁹,V¹⁰, V¹¹ and V¹² are selected from the following:

wherein:

T⁷ is absent and V⁷ is —NR¹⁵CO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is EDA and V¹¹ is —CO—; and

l is 0; or

wherein:

T⁷ is absent and V⁷ is —NR¹⁵CO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent; and

k and l are each 0; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is an amino acid analog and V⁹ is —NH—;

T¹⁰ is (PEG)_(n) and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent; and

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CONH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent; and

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is substituted (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is (C₁-C₁2)alkyl and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —O—;

T¹⁰ is (C₁-C₁2)alkyl and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent;

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is an amino acid analog and V⁸ is absent;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CONH—;

T⁹ is substituted (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is AA and V⁸ is —NH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PAP and V¹⁰ is —COO—; and

k and l are each 0.

In certain embodiments of the second linker L^(B), the left-hand side ofthe linker structure is attached to the hydrazinyl-indolyl or thehydrazinyl-pyrrolo-pyridinyl conjugation moiety, and the right-hand sideof the linker structure is attached to the camptothecine or acamptothecine derivative. In certain embodiments of the second linkerL^(B), the left-hand side of the linker structure is attached to thehydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl conjugationmoiety through a linking functional group, such as —NHCO-.

In certain embodiments, the conjugate is an antibody-drug conjugatewhere the antibody and the drug are linked together by a linker (e.g., Lor L^(B)), as described above. In some instances, the linker is acleavable linker. A cleavable linker is a linker that includes one ormore cleavable moieties, where the cleavable moiety includes one or morebonds that can dissociate under certain conditions, thus separating thecleavable linker into two or more separatable portions. For example, thecleavable moiety may include one or more covalent bonds, which undercertain conditions, can dissociate or break apart to separate thecleavable linker into two or more portions. As such a cleavable linkercan be included in an antibody-drug conjugate, such that underappropriate conditions, the cleavable linker is cleaved to separate orrelease the drug from the antibody at a desired target site of actionfor the drug.

In some instances, the cleavable linker includes two cleavable moieties,such as a first cleavable moiety and a second cleavable moiety. Thecleavable moieties can be configured such that cleavage of bothcleavable moieties is needed in order to separate or release the drugfrom the antibody at a desired target site of action for the drug. Forexample, cleavage of the cleavable linker can be achieved by initiallycleaving one of the two cleavable moieties and then cleaving the otherof the two cleavable moieties. In certain embodiments, the cleavablelinker includes a first cleavable moiety and a second cleavable moietythat hinders cleavage of the first cleavable moiety. By “hinderscleavage” is meant that the presence of an uncleaved second cleavablemoiety reduces the likelihood or substantially inhibits the cleavage ofthe first cleavable moiety, thus substantially reducing the amount orpreventing the cleavage of the cleavable linker. For instance, thepresence of uncleaved second cleavable moiety can hinder cleavage of thefirst cleavable moiety. The hinderance of cleavage of the firstcleavable moiety by the presence of the second cleavable moiety, inturn, substantially reduces the amount or prevents the release of thedrug from the antibody. For example, the premature release of the drugfrom the antibody can be substantially reduced or prevented until theantibody-drug conjugate is at or near the desired target site of actionfor the drug.

In some cases, since the second cleavable moiety hinders cleavage of thefirst cleavable moiety, cleavage of the cleavable linker can be achievedby initially cleaving the second cleavable moiety and then cleaving thefirst cleavable moiety. Cleavage of the second cleavable moiety canreduce or eliminate the hinderance on the cleavage of the firstcleavable moiety, thus allowing the first cleavable moiety to becleaved. Cleavage of the first cleavable moiety can result in thecleavable linker dissociating or separating into two or more portions asdescribed above to release the drug from the antibody-drug conjugate. Insome instances, cleavage of the first cleavable moiety does notsubstantially occur in the presence of an uncleaved second cleavablemoiety. By substantially is meant that about 10% or less cleavage of thefirst cleavable moiety occurs in the presence of an uncleaved secondcleavable moiety, such as about 9% or less, or about 8% or less, orabout 7% or less, or about 6% or less, or about 5% or less, or about 4%or less, or about 3% or less, or about 2% or less, or about 1% or less,or about 0.5% or less, or about 0.1% or less cleavage of the firstcleavable moiety occurs in the presence of an uncleaved second cleavablemoiety.

Stated another way, the second cleavable moiety can protect the firstcleavable moiety from cleavage. For instance, the presence of uncleavedsecond cleavable moiety can protect the first cleavable moiety fromcleavage, and thus substantially reduce or prevent premature release ofthe drug from the antibody until the antibody-drug conjugate is at ornear the desired target site of action for the drug. As such, cleavageof the second cleavable moiety exposes the first cleavable moiety (e.g.,deprotects the first cleavable moiety), thus allowing the firstcleavable moiety to be cleaved, which results in cleavage of thecleavable linker, which, in turn, separates or releases the drug fromthe antibody at a desired target site of action for the drug asdescribed above. In certain instances, cleavage of the second cleavablemoiety exposes the first cleavable moiety to subsequent cleavage, butcleavage of the second cleavable moiety does not in and of itself resultin cleavage of the cleavable linker (i.e., cleavage of the firstcleavable moiety is still needed in order to cleave the cleavablelinker).

The cleavable moieties included in the cleavable linker may each be anenzymatically cleavable moiety. For example, the first cleavable moietycan be a first enzymatically cleavable moiety and the second cleavablemoiety can be a second enzymatically cleavable moiety. An enzymaticallycleavable moiety is a cleavable moiety that can be separated into two ormore portions as described above through the enzymatic action of anenzyme. The enzymatically cleavable moiety can be any cleavable moietythat can be cleaved through the enzymatic action of an enzyme, such as,but not limited to, a peptide, a glycoside, and the like. In someinstances, the enzyme that cleaves the enzymatically cleavable moiety ispresent at a desired target site of action, such as the desired targetsite of action of the drug that is to be released from the antibody-drugconjugate. In some cases, the enzyme that cleaves the enzymaticallycleavable moiety is not present in a significant amount in other areas,such as in whole blood, plasma or serum. As such, the cleavage of anenzymatically cleavable moiety can be controlled such that substantialcleavage occurs at the desired site of action, whereas cleavage does notsignificantly occur in other areas or before the antibody-drug conjugatereaches the desired site of action.

For example, as described herein, antibody-drug conjugates of thepresent disclosure can be used for the treatment of cancer, such as forthe delivery of a cancer therapeutic drug to a desired site of actionwhere the cancer cells are present. In some cases, enzymes, such as theprotease enzyme cathepsin B, can be a biomarker for cancer that isoverexpressed in cancer cells. The overexpression, and thuslocalization, of certain enzymes in cancer can be used in the context ofthe enzymatically cleavable moieties included in the cleavable linkersof the antibody-drug conjugates of the present disclosure tospecifically release the drug at the desired site of action (i.e., thesite of the cancer (and overexpressed enzyme)). Thus, in someembodiments, the enzymatically cleavable moiety is a cleavable moiety(e.g., a peptide) that can be cleaved by an enzyme that is overexpressedin cancer cells. For instance, the enzyme can be the protease enzymecathepsin B. As such, in some instances, the enzymatically cleavablemoiety is a cleavable moiety (e.g., a peptide) that can be cleaved by aprotease enzyme, such as cathepsin B.

In certain embodiments, the enzymatically cleavable moiety is a peptide.The peptide can be any peptide suitable for use in the cleavable linkerand that can be cleaved through the enzymatic action of an enzyme.Non-limiting examples of peptides that can be used as an enzymaticallycleavable moiety include, for example, Val-Ala, Phe-Lys, and the like.For example, the first cleavable moiety described above (i.e., thecleavable moiety protected from premature cleavage by the secondcleavable moiety) can include a peptide. The presence of uncleavedsecond cleavable moiety can protect the first cleavable moiety (peptide)from cleavage by a protease enzyme (e.g., cathepsin B), and thussubstantially reduce or prevent premature release of the drug from theantibody until the antibody-drug conjugate is at or near the desiredtarget site of action for the drug. In some instances, one of the aminoacid residues of the peptide that comprises the first cleavable moietyis linked to or includes a substituent, where the substituent comprisesthe second cleavable moiety. In some instances, the second cleavablemoiety includes a glycoside.

In some embodiments, the enzymatically cleavable moiety is sugar moiety,such as a glycoside (or glyosyl). In some cases, the glycoside canfacilitate an increase in the hydrophilicity of the cleavable linker ascompared to a cleavable linker that does not include the glycoside. Theglycoside can be any glycoside or glycoside derivative suitable for usein the cleavable linker and that can be cleaved through the enzymaticaction of an enzyme. For example, the second cleavable moiety (i.e., thecleavable moiety that protects the first cleavable moiety from prematurecleavage) can be a glycoside. For instance, in some embodiments, thefirst cleavable moiety includes a peptide and the second cleavablemoiety includes a glycoside. In certain embodiments, the secondcleavable moiety is a glycoside or glycoside derivative selected from aglucuronide, a galactoside, a glucoside, a mannoside, a fucoside,O-GlcNAc, and O-GalNAc. In some instances, the second cleavable moietyis a glucuronide. In some instances, the second cleavable moiety is agalactoside. In some instances, the second cleavable moiety is aglucoside. In some instances, the second cleavable moiety is amannoside. In some instances, the second cleavable moiety is a fucoside.In some instances, the second cleavable moiety is O-GlcNAc. In someinstances, the second cleavable moiety is O-GalNAc.

The glycoside can be attached (covalently bonded) to the cleavablelinker through a glycosidic bond. The glycosidic bond can link theglycoside to the cleavable linker through various types of bonds, suchas, but not limited to, an O-glycosidic bond (an O-glycoside), anN-glycosidic bond (a glycosylamine), an S-glycosidic bond (athioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl). Insome instances, the glycosidic bond is an O-glycosidic bond (an0-glycoside). In some cases, the glycoside can be cleaved from thecleavable linker it is attached to by an enzyme (e.g., throughenzymatically-mediated hydrolysis of the glycosidic bond). A glycosidecan be removed or cleaved from the cleavable linker by any convenientenzyme that is able to carry out the cleavage (hydrolysis) of theglycosidic bond that attaches the glycoside to the cleavable linker. Anexample of an enzyme that can be used to mediate the cleavage(hydrolysis) of the glycosidic bond that attaches the glycoside to thecleavable linker is a glucuronidase, a glycosidase, such as agalactosidase, a glucosidase, a mannosidase, a fucosidase, and the like.Other suitable enzymes may also be used to mediate the cleavage(hydrolysis) of the glycosidic bond that attaches the glycoside to thecleavable linker. In some cases, the enzyme used to mediate the cleavage(hydrolysis) of the glycosidic bond that attaches the glycoside to thecleavable linker is found at or near the desired site of action for thedrug of the antibody-drug conjugate. For instance, the enzyme can be alysosomal enzyme, such as a lysosomal glycosidase, found in cells at ornear the desired site of action for the drug of the antibody-drugconjugate. In some cases, the enzyme is an enzyme found at or near thetarget site where the enzyme that mediates cleavage of the firstcleavable moiety is found.

In certain embodiments, the conjugate of formula (I) has a structureselected from the following:

Any of the chemical entities, linkers and coupling moieties set forth inthe structures above may be adapted for use in the subject compounds andconjugates.

Additional disclosure related to hydrazinyl-indolyl andhydrazinyl-pyrrolo-pyridinyl compounds and methods for producing aconjugate is found in U.S. Pat. Nos. 9,310,374 and 9,493,413, thedisclosures of each of which are incorporated herein by reference.Additional disclosure related to cleavable linkers is found in U.S.Provisional Application No. 63/116,632, filed Nov. 20, 2020, thedisclosure of which is incorporated herein by reference.

Compounds Useful for Producing Conjugates

The present disclosure provides hydrazinyl-indolyl andhydrazinyl-pyrrolo-pyridinyl compounds useful for producing theconjugates described herein. In certain embodiments, thehydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound may be aconjugation moiety useful for conjugation of a polypeptide (e.g., anantibody) and a drug or active agent (e.g., a camptothecine or acamptothecine derivative). For example, the hydrazinyl-indolyl orhydrazinyl-pyrrolo-pyridinyl compound may be bound to the polypeptide(antibody) and also bound to the drug or active agent, thus indirectlybinding the polypeptide (antibody) and the drug together.

In certain embodiments, the compound is a compound of formula (III):

wherein

Z is CR⁴ or N;

R⁸ and R⁹ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁸ and R⁹ are optionally cyclically linked to form a 5 or 6-memberedheterocyclyl;

each R¹⁰ is independently selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is a linker attached to a compound of formula (II) at R¹, R², R³, R⁴,R⁵ or R⁶:

R¹ and R² are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R¹ and R² are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring;

R³ and R⁴ are each independently selected from hydrogen, halo, hydroxy,amino, substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR³ and R⁴ are optionally cyclically linked to form a 5 or 6-memberedcycloalkyl or heterocyclyl ring;

R⁵ is selected from hydrogen, halo, hydroxy, amino, substituted amino,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

R⁶ is selected from OH and OC(O)R¹¹; and

R¹¹ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl,

wherein at least one R¹⁰ is optionally linked to a second compound offormula (II).

In some instances, the compound of formula (II) has the structure offormula (IIa):

wherein R³ is OH and L is attached at R⁶; or L is attached at R³ and R⁶is OH

In some instances, the compound of formula (II) has the structure offormula (IIb):

wherein R^(1a) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1a) and R⁶ is OH.

In some instances, the compound of formula (II) has the structure offormula (IIc):

wherein R^(1b) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1b) and R⁶ is OH.

In some instances, the compound of formula (II) has the structure offormula (IId):

wherein R^(2a) and R^(2b) are each independently selected from H, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl,and L is attached at R⁶; or L is attached at R^(2a) or R^(2b) and R⁶ isOH.

In some instances, the compound of formula (II) has the structure offormula (IIe):

wherein R^(2c) is selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxylester, acyl, and sulfonyl, and attachment to L is indicated by the wavyline.

The substituents related to compounds of formula (II), (IIa), (IIb),(IIc), (IId) and (IIe) are described above. References to formula (II)are intended to also encompass formulae (IIa), (IIb), (IIc), (IId) and(IIe).

Regarding compounds of formula (III), the substituents Z, R⁸, R⁹, R¹⁰,L, L^(B), and W are as described above in relation to the conjugates offormula (I). Similarly, regarding the first linker L and the secondlinker L^(B) of formula (III), the T¹, T², T³, T⁴, T⁵, T⁶, V¹, V², V³,V⁴, V⁵ and V⁶, and T⁷, T⁸, T⁹, T¹⁰, T¹¹, T¹², V⁷, V⁸, V⁹, V¹⁰, V¹¹ andV¹² substituents are as described above in relation to the conjugates offormula (I).

For example, in some instances, T¹, T², T³, T⁴, T⁵ and T⁶ and V¹, V²,V³, V⁴, V⁵ and V⁶ are selected from the following:

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

T⁶ is EDA and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is absent and V⁵ is —NR¹⁵(C₆H₄)—; and

T⁶ is absent and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is —NR¹⁵—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —CO—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent;

T⁴ is EDA and V⁴ is —CO—; and

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is absent;

T³ is PABC and V³ is absent; and

d, e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABA and V⁵ is —CO—; and

T⁶ is (C₁-C₁₂)alkyl and V⁶ is —SO₂—; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is substituted (C₁-C₁₂)alkyl and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

T⁵ is (C₁-C₁₂)alkyl and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is 4AP and V² is —CO—;

T³ is (C₁-C₁₂)alkyl and V³ is —O—;

T⁴ is (C₁-C₁₂)alkyl and V⁴ is —CO—;

T⁵ is AA and V⁵ is absent;

T⁶ is PABC and V⁶ is absent; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is absent;

T³ is AA and V³ is absent;

T⁴ is PABC and V⁴ is absent;

e and f are each 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CONH—;

T³ is substituted (C₁-C₁₂)alkyl and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is AA and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABC and V⁵ is absent;

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PABO and V⁵ is absent; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—;

T² is an amino acid analog and V² is —NH—;

T³ is (PEG)_(n) and V³ is —CO—;

T⁴ is AA and V⁴ is absent;

T⁵ is PAP and V⁵ is —COO—; and

f is 0; or

wherein:

T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—;

T² is (PEG)_(n) and V² is —CO—;

T³ is AA and V³ is absent;

T⁴ is PAP and V⁴ is —COO—; and

e and f are each 0.

For example, in some instances, T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² and V⁷, V⁸,V⁹, V¹⁰, V¹¹ and V¹² are selected from the following:

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is EDA and V¹¹ is —CO—; and

l is 0; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent; and

k and l are each 0; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is an amino acid analog and V⁹ is —NH—;

T¹⁰ is (PEG)_(n) and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent; and

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is absent and V⁷ is —NHCO—;

T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CONH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent; and

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is substituted (C₁-C₁₂)alkyl and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

T¹¹ is (C₁-C₁2)alkyl and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is 4AP and V⁸ is —CO—;

T⁹ is (C₁-C₁₂)alkyl and V⁹ is —O—;

T¹⁰ is (C₁-C₁2)alkyl and V¹⁰ is —CO—;

T¹¹ is AA and V¹¹ is absent;

T¹² is PABC and V¹² is absent; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is an amino acid analog and V⁸ is absent;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PABC and V¹⁰ is absent;

k and l are each 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CONH—;

T⁹ is substituted (C₁-C₁₂)alkyl and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;

T⁸ is AA and V⁸ is —NH—;

T⁹ is (PEG)_(n) and V⁹ is —CO—;

T¹⁰ is AA and V¹⁰ is absent;

T¹¹ is PABC and V¹¹ is absent;

l is 0; or

wherein:

T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—;

T⁸ is (PEG)_(n) and V⁸ is —CO—;

T⁹ is AA and V⁹ is absent;

T¹⁰ is PAP and V¹⁰ is —COO—; and

k and l are each 0.

Compounds of formula (III) can be used in conjugation reactionsdescribed herein, where a drug or active agent attached to ahydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moietyis conjugated to a polypeptide (e.g., antibody) to form an antibody-drugconjugate.

In certain embodiments, the compound of formula (III) has the followingstructure:

Any of the chemical entities, linkers and conjugation moieties set forthin the structures above may be adapted for use in the subject compoundsand conjugates.

Polypeptides and Antibodies

As noted above, a subject conjugate can comprise as substituent W apolypeptide (e.g., an antibody). The amino acid sequence of thepolypeptide (antibody) has been modified to include a 2-formylglycine(fGly) residue. As used herein, amino acids may be referred to by theirstandard name, their standard three letter abbreviation and/or theirstandard one letter abbreviation, such as: Alanine or Ala or A; Cysteineor Cys or C; Aspartic acid or Asp or D; Glutamic acid or Glu or E;Phenylalanine or Phe or F; Glycine or Gly or G; Histidine or His or H;Isoleucine or Ile or I; Lysine or Lys or K; Leucine or Leu or L;Methionine or Met or M; Asparagine or Asn or N; Proline or Pro or P;Glutamine or Gln or Q; Arginine or Arg or R; Serine or Ser or S;Threonine or Thr or T; Valine or Val or V; Tryptophan or Trp or W; andTyrosine or Tyr or Y.

In certain embodiments, the amino acid sequence of the polypeptide(antibody) is modified to include a sulfatase motif that contains aserine or cysteine residue that is capable of being converted (oxidized)to a 2-formylglycine (fGly) residue by action of a formylglycinegenerating enzyme (FGE) either in vivo (e.g., at the time of translationof an aldehyde tag-containing protein in a cell) or in vitro (e.g., bycontacting an aldehyde tag-containing protein with an FGE in a cell-freesystem). Such sulfatase motifs may also be referred to herein as anFGE-modification site.

Sulfatase Motifs

A minimal sulfatase motif of an aldehyde tag is usually 5 or 6 aminoacid residues in length, usually no more than 6 amino acid residues inlength. Sulfatase motifs provided in an Ig polypeptide are at least 5 or6 amino acid residues, and can be, for example, from 5 to 16, 6-16,5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10,5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define asulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6amino acid residues in length.

In certain embodiments, polypeptides of interest include those where oneor more amino acid residues, such as 2 or more, or 3 or more, or 4 ormore, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 ormore, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or19 or more, or 20 or more amino acid residues have been inserted,deleted, substituted (replaced) relative to the native amino acidsequence to provide for a sequence of a sulfatase motif in thepolypeptide. In certain embodiments, the polypeptide includes amodification (insertion, addition, deletion, and/orsubstitution/replacement) of less than 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues of the aminoacid sequence relative to the native amino acid sequence of thepolypeptide. Where an amino acid sequence native to the polypeptide(e.g., antibody) contains one or more residues of the desired sulfatasemotif, the total number of modifications of residues can be reduced,e.g., by site-specification modification (insertion, addition, deletion,substitution/replacement) of amino acid residues flanking the nativeamino acid residues to provide a sequence of the desired sulfatasemotif. In certain embodiments, the extent of modification of the nativeamino acid sequence of the target antibody is minimized, so as tominimize the number of amino acid residues that are inserted, deleted,substituted (replaced), or added (e.g., to the N- or C-terminus).Minimizing the extent of amino acid sequence modification of the targetantibody may minimize the impact such modifications may have uponantibody function and/or structure.

It should be noted that while aldehyde tags of particular interest arethose comprising at least a minimal sulfatase motif (also referred to a“consensus sulfatase motif”), it will be readily appreciated that longeraldehyde tags are both contemplated and encompassed by the presentdisclosure and can find use in the compositions and methods of thepresent disclosure. Aldehyde tags can thus comprise a minimal sulfatasemotif of 5 or 6 residues, or can be longer and comprise a minimalsulfatase motif which can be flanked at the N- and/or C-terminal sidesof the motif by additional amino acid residues. Aldehyde tags of, forexample, 5 or 6 amino acid residues are contemplated, as well as longeramino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more amino acid residues.

An aldehyde tag can be present at or near the C-terminus of an Ig heavychain; e.g., an aldehyde tag can be present within 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids of the C-terminus of a native, wild-type Igheavy chain. An aldehyde tag can be present within a CH1 domain of an Igheavy chain. An aldehyde tag can be present within a CH2 domain of an Igheavy chain. An aldehyde tag can be present within a CH3 domain of an Igheavy chain. An aldehyde tag can be present in an Ig light chainconstant region, e.g., in a kappa light chain constant region or alambda light chain constant region.

In certain embodiments, the sulfatase motif used may be described by theformula:

X¹Z¹⁰X²Z²⁰X³Z³⁰  (I′)

where

Z¹⁰ is cysteine or serine (which can also be represented by (C/S));

Z²⁰ is either a proline or alanine residue (which can also berepresented by (P/A));

Z³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) orhistidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A),glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P),e.g., A, G, L, V, or I;

X¹ is present or absent and, when present, can be any amino acid, e.g.,an aliphatic amino acid, a sulfur-containing amino acid, or a polar,uncharged amino acid, (i.e., other than an aromatic amino acid or acharged amino acid), e.g., L, M, V, S or T, e.g., L, M, S or V, with theproviso that when the sulfatase motif is at the N-terminus of the targetpolypeptide, XV is present; and

X² and X³ independently can be any amino acid, though usually analiphatic amino acid, a polar, uncharged amino acid, or a sulfurcontaining amino acid (i.e., other than an aromatic amino acid or acharged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

The amino acid sequence of an antibody heavy and/or light chain can bemodified to provide a sequence of at least 5 amino acids of the formulaX¹Z¹⁰X²Z²⁰X³Z³⁰, where

Z¹⁰ is cysteine or serine;

Z²⁰ is a proline or alanine residue;

Z³⁰ is an aliphatic amino acid or a basic amino acid;

X¹ is present or absent and, when present, is any amino acid, with theproviso that when the heterologous sulfatase motif is at an N-terminusof the polypeptide, X¹ is present;

X² and X³ are each independently any amino acid.

The sulfatase motif is generally selected so as to be capable ofconversion by a selected FGE, e.g., an FGE present in a host cell inwhich the aldehyde tagged polypeptide is expressed or an FGE which is tobe contacted with the aldehyde tagged polypeptide in a cell-free invitro method.

For example, where the FGE is a eukaryotic FGE (e.g., a mammalian FGE,including a human FGE), the sulfatase motif can be of the formula:

X¹CX²PX³Z³⁰  (I″)

where

X¹ may be present or absent and, when present, can be any amino acid,e.g., an aliphatic amino acid, a sulfur-containing amino acid, or apolar, uncharged amino acid, (i.e., other than an aromatic amino acid ora charged amino acid), e.g., L, M, S or V, with the proviso that whenthe sulfatase motif is at the N-terminus of the target polypeptide, X¹is present;

X² and X³ independently can be any amino acid, e.g., an aliphatic aminoacid, a sulfur-containing amino acid, or a polar, uncharged amino acid,(i.e., other than an aromatic amino acid or a charged amino acid), e.g.,S, T, A, V, G, or C, e.g., S, T, A, V or G; and

Z³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) orhistidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A),glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P),e.g., A, G, L, V, or I.

Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO://),MCTPSR (SEQ ID NO://), VCTPSR (SEQ ID NO://), LCSPSR (SEQ ID NO://),LCAPSR (SEQ ID NO://), LCVPSR (SEQ ID NO://), LCGPSR (SEQ ID NO://),ICTPAR (SEQ ID NO://), LCTPSK (SEQ ID NO://), MCTPSK (SEQ ID NO://),VCTPSK (SEQ ID NO://), LCSPSK (SEQ ID NO://), LCAPSK (SEQ ID NO://),LCVPSK (SEQ ID NO://), LCGPSK (SEQ ID NO://), LCTPSA (SEQ ID NO://),ICTPAA (SEQ ID NO://), MCTPSA (SEQ ID NO://), VCTPSA (SEQ ID NO://),LCSPSA (SEQ ID NO://), LCAPSA (SEQ ID NO://), LCVPSA (SEQ ID NO://), andLCGPSA (SEQ ID NO://).

fGly-Containing Sequences

Upon action of FGE on the antibody heavy and/or light chain, the serineor the cysteine in the sulfatase motif is modified to fGly. Thus, thefGly-containing sulfatase motif can be of the formula:

X¹(fGly)X²Z²⁰X³Z³⁰  (I′″)

where

fGly is the formylglycine residue;

Z²⁰ is either a proline or alanine residue (which can also berepresented by (P/A));

Z³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) orhistidine (H), usually lysine), or an aliphatic amino acid (alanine (A),glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P),e.g., A, G, L, V, or I;

X¹ may be present or absent and, when present, can be any amino acid,e.g., an aliphatic amino acid, a sulfur-containing amino acid, or apolar, uncharged amino acid, (i.e., other than an aromatic amino acid ora charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with theproviso that when the sulfatase motif is at the N-terminus of the targetpolypeptide, X¹ is present; and

X² and X³ independently can be any amino acid, e.g., an aliphatic aminoacid, a sulfur-containing amino acid, or a polar, uncharged amino acid,(i.e., other than an aromatic amino acid or a charged amino acid), e.g.,S, T, A, V, G or C, e.g., S, T, A, V or G.

As described above, to produce the conjugate, the polypeptide containingthe fGly residue may be conjugated to a drug or active agent by reactionof the fGly with a reactive moiety (e.g., a hydrazinyl-indolyl or ahydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above) ofa linker attached to the drug or active agent to produce anfGly′-containing sulfatase motif. As used herein, the term fGly′ refersto the amino acid residue of the sulfatase motif that is coupled to thedrug or active agent through a linker, as described herein. Thus, thefGly′-containing sulfatase motif can be of the formula:

X¹(fGly′)X²Z²⁰X³Z³⁰  (II)

where

fGly′ is the amino acid residue coupled to the drug or active agentthrough a linker as described herein;

Z²⁰ is either a proline or alanine residue (which can also berepresented by (P/A));

Z³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) orhistidine (H), usually lysine), or an aliphatic amino acid (alanine (A),glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P),e.g., A, G, L, V, or I;

X¹ may be present or absent and, when present, can be any amino acid,e.g., an aliphatic amino acid, a sulfur-containing amino acid, or apolar, uncharged amino acid, (i.e., other than an aromatic amino acid ora charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with theproviso that when the sulfatase motif is at the N-terminus of the targetpolypeptide, XV is present; and

X² and X³ independently can be any amino acid, e.g., an aliphatic aminoacid, a sulfur-containing amino acid, or a polar, uncharged amino acid,(i.e., other than an aromatic amino acid or a charged amino acid), e.g.,S, T, A, V, G or C, e.g., S, T, A, V or G.

Site of Modification

As noted above, the amino acid sequence of the polypeptide (antibody) ismodified to include a sulfatase motif that contains a serine or cysteineresidue that is capable of being converted (oxidized) to an fGly residueby action of an FGE either in vivo (e.g., at the time of translation ofan aldehyde tag-containing protein in a cell) or in vitro (e.g., bycontacting an aldehyde tag-containing protein with an FGE in a cell-freesystem). The antibody used to generate a conjugate of the presentdisclosure include at least an Ig constant region, e.g., an Ig heavychain constant region (e.g., at least a CH1 domain; at least a CH1 and aCH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, anda CH4 domain), or an Ig light chain constant region. Such Igpolypeptides are referred to herein as “target Ig polypeptides” or“target antibodies”.

The site in an antibody into which a sulfatase motif is introduced canbe any convenient site. As noted above, in some instances, the extent ofmodification of the native amino acid sequence of the target polypeptideis minimized, so as to minimize the number of amino acid residues thatare inserted, deleted, substituted (replaced), and/or added (e.g., tothe N- or C-terminus). Minimizing the extent of amino acid sequencemodification of the target antibody may minimize the impact suchmodifications may have upon antibody function and/or structure.

An antibody heavy chain constant region can include Ig constant regionsof any heavy chain isotype, non-naturally occurring Ig heavy chainconstant regions (including consensus Ig heavy chain constant regions).An Ig constant region amino acid sequence can be modified to include analdehyde tag, where the aldehyde tag is present in or adjacent asolvent-accessible loop region of the Ig constant region. An Ig constantregion amino acid sequence can be modified by insertion and/orsubstitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16amino acids, or more than 16 amino acids, to provide an amino acidsequence of a sulfatase motif as described above.

In some cases, an aldehyde-tagged antibody comprises an aldehyde-taggedIg heavy chain constant region (e.g., at least a CH1 domain; at least aCH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, aCH3, and a CH4 domain). The aldehyde-tagged Ig heavy chain constantregion can include heavy chain constant region sequences of an IgA, IgM,IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypicvariant of same, e.g., human heavy chain constant region sequences ormouse heavy chain constant region sequences, a hybrid heavy chainconstant region, a synthetic heavy chain constant region, or a consensusheavy chain constant region sequence, etc., modified to include at leastone sulfatase motif that can be modified by an FGE to generate anfGly-modified Ig polypeptide. Allotypic variants of Ig heavy chains areknown in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4.

In some cases, an aldehyde-tagged antibody comprises an aldehyde-taggedIg light chain constant region. The aldehyde-tagged Ig light chainconstant region can include constant region sequences of a kappa lightchain, a lambda light chain, e.g., human kappa or lambda light chainconstant regions, a hybrid light chain constant region, a syntheticlight chain constant region, or a consensus light chain constant regionsequence, etc., that includes at least one sulfatase motif that can bemodified by an FGE to generate an fGly-modified antibody. Exemplaryconstant regions include human gamma 1 and gamma 3 regions. With theexception of the sulfatase motif, a modified constant region may have awild-type amino acid sequence, or it may have an amino acid sequencethat is at least 70% identical (e.g., at least 80%, at least 90% or atleast 95% identical) to a wild type amino acid sequence.

In some embodiments the sulfatase motif is at a position other than, orin addition to, the C-terminus of the Ig polypeptide heavy chain. Asnoted above, an isolated aldehyde-tagged antibody can comprise a heavychain constant region amino acid sequence modified to include asulfatase motif as described above, where the sulfatase motif is in oradjacent a surface-accessible loop region of the antibody heavy chainconstant region.

A sulfatase motif can be provided within or adjacent one or more ofthese amino acid sequences of such modification sites of an Ig heavychain. For example, an Ig heavy chain polypeptide amino acid sequencecan be modified (e.g., where the modification includes one or more aminoacid residue insertions, deletions, and/or substitutions) at one or moreof these amino acid sequences to provide a sulfatase motif adjacent andN-terminal and/or adjacent and C-terminal to these modification sites.Alternatively or in addition, an Ig heavy chain polypeptide amino acidsequence can be modified (e.g., where the modification includes one ormore amino acid residue insertions, deletions, and/or substitutions) atone or more of these amino acid sequences to provide a sulfatase motifbetween any two residues of the Ig heavy chain modifications sites. Insome embodiments, an Ig heavy chain polypeptide amino acid sequence maybe modified to include two motifs, which may be adjacent to one another,or which may be separated by one, two, three, four or more (e.g., fromabout 1 to about 25, from about 25 to about 50, or from about 50 toabout 100, or more, amino acids. Alternatively or in addition, where anative amino acid sequence provides for one or more amino acid residuesof a sulfatase motif sequence, selected amino acid residues of themodification sites of an Ig heavy chain polypeptide amino acid sequencecan be modified (e.g., where the modification includes one or more aminoacid residue insertions, deletions, and/or substitutions) so as toprovide a sulfatase motif at the modification site.

An antibody used in an antibody-drug conjugate of the present disclosurecan have any of a variety of antigen-binding specificities, includingbut not limited to, e.g., an antigen present on a cancer cell; anantigen present on an autoimmune cell; an antigen present on apathogenic microorganism; an antigen present on a virus-infected cell(e.g., a human immunodeficiency virus-infected cell); an antigen presenton a diseased cell; and the like. For example, an antibody conjugate canbind an antigen, where the antigen is present on the surface of thecell. An antibody conjugate of the present disclosure can bind antigenwith a suitable binding affinity, e.g., from 5×10⁻⁶ M to 10⁻⁷ M, from10⁻⁷ M to 5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from 10⁻⁸ M to 5×10⁻⁸ M,from 5×10⁻⁸ M to 10⁻⁹ M, or a binding affinity greater than 10⁻⁹ M.

As non-limiting examples, a subject antibody conjugate can bind anantigen present on a cancer cell (e.g., a tumor-specific antigen; anantigen that is over-expressed on a cancer cell; etc.), and theconjugated moiety can be a drug, such as a cytotoxic compound (e.g., acytotoxic small molecule, a cytotoxic synthetic peptide, etc.). Forexample, a subject antibody conjugate can be specific for an antigen ona cancer cell, where the conjugated moiety is a drug, such as acytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxicsynthetic peptide, etc.).

As further non-limiting examples, a subject antibody conjugate can bindan antigen present on a cell infected with a virus (e.g., where theantigen is encoded by the virus; where the antigen is expressed on acell type that is infected by a virus; etc.), and the conjugated moietycan be a drug, such as a viral fusion inhibitor. For example, a subjectantibody conjugate can bind an antigen present on a cell infected with avirus, and the conjugated moiety can be a drug, such as a viral fusioninhibitor.

Drugs for Conjugation to a Polypeptide

The present disclosure provides drug-polypeptide conjugates (e.g.,antibody-drug conjugates). Drugs suitable for use, or that can bemodified to be rendered suitable for use, as a reactive partner toconjugate to a polypeptide (e.g., an antibody) as described hereininclude a camptothecine or a camptothecine derivative. For example,camptothecine and camptothecine derivatives suitable for use in theconjugates and compounds described herein include, by are not limited tocompounds of formula (II), (IIa), (IIb), (IIc) and (IId), as describedabove.

Embodiments of the present disclosure include conjugates where apolypeptide (e.g., an antibody) is conjugated to one or more drugmoieties, such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drugmoieties, or 10 or more drug moieties. The drug moieties may beconjugated to the antibody at one or more sites in the polypeptide(antibody), as described herein. In certain embodiments, the conjugateshave an average drug-to-antibody ratio (DAR) (molar ratio) in the rangeof from 0.1 to 10, or from 0.5 to 10, or from 1 to 10, such as from 1 to9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, orfrom 1 to 4, or from 1 to 3, or from 1 to 2. In certain embodiments, theconjugates have an average DAR from 1 to 3, such as 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9 or 3. In certain embodiments, the conjugates have an average DAR of1 to 2. In certain embodiments, the conjugates have an average DAR of 2to 3. By average is meant the arithmetic mean.

Formulations

The conjugates of the present disclosure can be formulated in a varietyof different ways. In general, where the conjugate is an antibody-drugconjugate, the conjugate is formulated in a manner compatible with thedrug, the antibody, the condition to be treated, and the route ofadministration to be used.

In some embodiments, provided is a pharmaceutical composition thatincludes any of the conjugates of the present disclosure and apharmaceutically-acceptable excipient.

The conjugate (e.g., antibody-drug conjugate) can be provided in anysuitable form, e.g., in the form of a pharmaceutically acceptable salt,and can be formulated for any suitable route of administration, e.g.,oral, topical or parenteral administration. Where the conjugate isprovided as a liquid injectable (such as in those embodiments where theyare administered intravenously or directly into a tissue), the conjugatecan be provided as a ready-to-use dosage form, or as a reconstitutablestorage-stable powder or liquid composed of pharmaceutically acceptablecarriers and excipients.

Methods for formulating conjugates can be adapted from those readilyavailable. For example, conjugates can be provided in a pharmaceuticalcomposition comprising a therapeutically effective amount of a conjugateand a pharmaceutically acceptable carrier (e.g., saline). Thepharmaceutical composition may optionally include other additives (e.g.,buffers, stabilizers, preservatives, and the like). In some embodiments,the formulations are suitable for administration to a mammal, such asthose that are suitable for administration to a human.

Methods of Treatment

The antibody-drug conjugates of the present disclosure find use intreatment of a condition or disease in a subject that is amenable totreatment by administration of the parent drug (i.e., the camptothecineor camptothecine derivative prior to conjugation to the antibody).

In some embodiments, provided are methods that include administering toa subject an effective amount (e.g., a therapeutically effective amount)of any of the conjugates of the present disclosure.

In certain aspects, provided are methods of delivering a drug to atarget site in a subject, the method including administering to thesubject a pharmaceutical composition including any of the conjugates ofthe present disclosure, where the administering is effective to releasea therapeutically effective amount of the drug (e.g., a camptothecine ora camptothecine derivative) from the conjugate at the target site in thesubject. For example, as described herein, antibody-drug conjugates ofthe present disclosure can include a cleavable linker, such as anenzymatically cleavable linker that includes a first enzymaticallycleavable moiety and a second enzymatically cleavable moiety. In someinstances, the cleavable linker can be cleaved under appropriateconditions to separate or release the drug from the antibody at adesired target site of action for the drug. For example, the secondcleavable moiety, which protects the first cleavable moiety fromcleavage, may be cleaved in order to allow the first cleavable moiety tobe cleaved, which results in cleavage of the cleavable linker into twoor more portions, thus releasing the drug from the antibody-drugconjugate at a desired site of action.

In certain embodiments, the first cleavable moiety can be anenzymatically cleavable moiety. In some instances, the enzyme thatfacilitates cleavage of the first cleavable moiety is an enzyme that isadministered to the subject to be treated (i.e., exogenous to thesubject to be treated). For example, a first enzyme can be administeredbefore, concurrently with, or after administration of an antibody-drugconjugate described herein.

In certain embodiments, the second cleavable moiety can be anenzymatically cleavable moiety. In some instances, the enzyme thatfacilitates cleavage of the second cleavable moiety is an enzyme that isadministered to the subject to be treated (i.e., exogenous to thesubject to be treated). For example, a second enzyme can be administeredbefore, concurrently with, or after administration of an antibody-drugconjugate described herein. In certain embodiments, the first enzyme andthe second enzyme are different enzymes.

In other instances, the first enzyme that facilitates cleavage of thefirst cleavable moiety is an enzyme that is present in the subject to betreated (i.e., endogenous to the subject to be treated). For instance,the first enzyme may be present at the desired site of action for thedrug of the antibody-drug conjugate. The antibody of the antibody-drugconjugate may be specifically targeted to a desired site of action(e.g., may specifically bind to an antigen present at a desired site ofaction), where the desired site of action also includes the presence ofthe first enzyme. In some instances, the first enzyme is present in anoverabundance at the desired site of action as compared to other areasin the body of the subject to be treated. For example, the first enzymemay be overexpressed at the desired site of action as compared to otherareas in the body of the subject to be treated. In some instances, thefirst enzyme is present in an overabundance at the desired site ofaction due to localization of the first enzyme at a particular area orlocation. For instance, the first enzyme may be associated with acertain structure within the desired site of action, such as lysosomes.In some cases, the first enzyme is present in an overabundance inlysosomes as compared to other areas in the body of the subject. In someembodiments, the lysosomes that include the first enzyme, are found at adesired site of action for the drug of the antibody-drug conjugate, suchas the site of a cancer or tumor that is to be treated with the drug. Incertain embodiments, the first enzyme is a protease, such as a humanprotease enzyme (e.g., cathepsin B).

In certain embodiments, the second enzyme that facilitates cleavage ofthe second cleavable moiety is an enzyme that is present in the subjectto be treated (i.e., endogenous to the subject to be treated). Forinstance, the second enzyme may be present at the desired site of actionfor the drug of the antibody-drug conjugate. The antibody of theantibody-drug conjugate may be specifically targeted to a desired siteof action (e.g., may specifically bind to an antigen present at adesired site of action), where the desired site of action also includesthe presence of the second enzyme. In some instances, the second enzymeis present in an overabundance at the desired site of action as comparedto other areas in the body of the subject to be treated. For example,the second enzyme may be overexpressed at the desired site of action ascompared to other areas in the body of the subject to be treated. Insome instances, the second enzyme is present in an overabundance at thedesired site of action due to localization of the second enzyme at aparticular area or location. For instance, the second enzyme may beassociated with a certain structure within the desired site of action,such as lysosomes. In some cases, the second enzyme is present in anoverabundance in lysosomes as compared to other areas in the body of thesubject. In some embodiments, the lysosomes that include the secondenzyme, are found at a desired site of action for the drug of theantibody-drug conjugate, such as the site of a cancer or tumor that isto be treated with the drug. In certain embodiments, the second enzymeis a glycosidase, such as a galactosidase, a glucosidase, or amannosidase.

Any suitable enzymes can be used for cleavage of the first cleavablemoiety and the second cleavable moiety of the antibody-drug conjugatesdescribed herein. Other enzymes may also be suitable for use in cleavageof the first cleavable moiety and the second cleavable moiety of theantibody-drug conjugates described herein, such as but not limited to,enzymes from other vertebrates (e.g., primates, mice, rats, cats, pigs,quails, goats, dogs, rabbits, etc.).

In certain embodiments, the antibody-drug conjugate is substantiallystable under standard conditions. By substantially stable is meant thatthe cleavable linker of the antibody-drug conjugate does not undergo asignificant amount of cleavage in the absence of a first enzyme and asecond enzyme as described above. For example, as described above, thesecond cleavable moiety can protect the first cleavable moiety frombeing cleaved, and as such the cleavable linker of the antibody-drugconjugate does not undergo a significant amount of cleavage in theabsence of a second enzyme as described above. For instance, thecleavable linker of the antibody-drug conjugate may be substantiallystable such that 25% or less of the antibody-drug conjugate is cleavedin the absence of the first enzyme and/or second enzyme, such as 20% orless, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or3% or less, or 2% or less, or 1% or less. In some cases, theantibody-drug conjugate is substantially stable such that the cleavablelinker of the antibody-drug conjugate does not undergo a significantamount of cleavage in the absence of the first enzyme and/or secondenzyme, but can be cleaved when in the presence of the first enzyme andthe second enzyme. For example, the antibody-drug conjugate can besubstantially stable after administration to a subject. In some cases,the antibody-drug conjugate is substantially stable after administrationto a subject, and then, when the antibody-drug conjugate is in thepresence of the second enzyme at a desired site of action, the secondcleavable moiety can be cleaved from the cleavable linker, thus exposingthe first cleavable moiety to subsequent cleavage by the first enzyme,which in turn releases the drug at the desired site of action. Incertain embodiments, after administration to a subject the antibody-drugconjugate is stable for an extended period of time in the absence of thefirst enzyme and/or second enzyme, such as 1 hr or more, or 2 hrs ormore, or 3 hrs or more, or 4 hrs or more, or 5 hrs or more, or 6 hrs ormore, or 7 hrs or more, or 8 hrs or more, or 9 hrs or more, or 10 hrs ormore, or 15 hrs or more, or 20 hrs or more, or 24 hrs (1 day) or more,or 2 days or more, or 3 days or more, or 4 days or more, or 5 days ormore, or 6 days or more, or 7 days (1 week) or more. In certainembodiments, the antibody-drug conjugate is stable at a range pH valuesfor an extended period of time in the absence of the first enzyme and/orsecond enzyme, such as at a pH ranging from 2 to 10, or from 3 to 9, orfrom 4 to 8, or from 5 to 8, or from 6 to 8, or from 7 to 8.

As described above, the antibody-drug conjugates of the presentdisclosure find use in treatment of a condition or disease in a subjectthat is amenable to treatment by administration of the parent drug. By“treatment” is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g., prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from thecondition, or at least the symptoms that characterize the condition.Thus treatment includes: (i) prevention, that is, reducing the risk ofdevelopment of clinical symptoms, including causing the clinicalsymptoms not to develop, e.g., preventing disease progression to aharmful state; (ii) inhibition, that is, arresting the development orfurther development of clinical symptoms, e.g., mitigating or completelyinhibiting an active disease; and/or (iii) relief, that is, causing theregression of clinical symptoms.

The subject to be treated can be one that is in need of therapy, wherethe subject to be treated is one amenable to treatment using the parentdrug. Accordingly, a variety of subjects may be amenable to treatmentusing the antibody-drug conjugates disclosed herein. Generally, suchsubjects are “mammals”, with humans being of interest. Other subjectscan include domestic pets (e.g., dogs and cats), livestock (e.g., cows,pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs,and rats, e.g., as in animal models of disease), as well as non-humanprimates (e.g., chimpanzees and monkeys).

The amount of antibody-drug conjugate administered can be initiallydetermined based on guidance of a dose and/or dosage regimen of theparent drug. In general, the antibody-drug conjugates can provide fortargeted delivery and/or enhanced serum half-life of the bound drug,thus providing for at least one of reduced dose or reducedadministrations in a dosage regimen. Thus, the antibody-drug conjugatescan provide for reduced dose and/or reduced administration in a dosageregimen relative to the parent drug prior to being conjugated in anantibody-drug conjugate of the present disclosure.

Furthermore, as noted above, because the antibody-drug conjugates canprovide for controlled stoichiometry of drug delivery, dosages ofantibody-drug conjugates can be calculated based on the number of drugmolecules provided on a per antibody-drug conjugate basis.

In some embodiments, multiple doses of an antibody-drug conjugate areadministered. The frequency of administration of an antibody-drugconjugate can vary depending on any of a variety of factors, e.g.,severity of the symptoms, condition of the subject, etc. For example, insome embodiments, an antibody-drug conjugate is administered once permonth, twice per month, three times per month, every other week, onceper week (qwk), twice per week, three times per week, four times perweek, five times per week, six times per week, every other day, daily(qd/od), twice a day (bds/bid), or three times a day (tds/tid), etc.

Methods of Treating Cancer

The present disclosure provides methods that include delivering aconjugate of the present disclosure to an individual having a cancer.The methods are useful for treating a wide variety of cancers,including, but not limited to breast, ovarian, colon, lung, stomach, andpancreatic cancer. In the context of cancer, the term “treating”includes one or more (e.g., each) of: reducing growth of a solid tumor,inhibiting replication of cancer cells, reducing overall tumor burden,and ameliorating one or more symptoms associated with a cancer.

Carcinomas that can be treated using a subject method include, but arenot limited to, colon carcinoma, colorectal carcinoma, gastriccarcinoma, lung carcinoma, including small cell carcinoma and non-smallcell carcinoma of the lung, pancreatic carcinoma, breast carcinoma,ovarian carcinoma, prostate carcinoma, adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, cervical carcinoma, uterine carcinoma, testicularcarcinoma, and epithelial carcinoma, etc.

In certain aspects, provided are methods of treating cancer in asubject, such methods including administering to the subject atherapeutically effective amount of a pharmaceutical compositionincluding any of the conjugates of the present disclosure, where theadministering is effective to treat cancer in the subject.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. By “average” is meant the arithmeticmean. Standard abbreviations may be used, e.g., bp, base pair(s); kb,kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h orhr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt,nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any purificationprotocol known in the art, including chromatography, such as HPLC,preparative thin layer chromatography, flash column chromatography andion exchange chromatography. Any suitable stationary phase can be used,including normal and reversed phases as well as ionic resins. In certainembodiments, the disclosed compounds are purified via silica gel and/oralumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4′ edition, Vol. 15/l, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

The subject compounds can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. A varietyof examples of synthetic routes that can be used to synthesize thecompounds disclosed herein are described in the schemes below.

Example 1 Materials and Methods General

Synthetic reagents were purchased from Sigma-Aldrich, Acros, AKScientific, or other commercial sources and were used withoutpurification. Anhydrous solvents were obtained from commercial sourcesin sealed bottles. Compounds 7, 13, 21, 28, 74, and 152, as well as HIPSlinkers 10, 18, and 36 were obtained commercially from ShanghaiMedicilon and used without purification. Compounds 156, 157, 169, and171 were purchased from other commercial sources; synthesis of compounds87 and 93 was previously reported. In all cases, solvent was removedunder reduced pressure with a Buchi Rotovapor R-114 equipped with aBuchi V-700 vacuum pump. Column chromatography was performed with aBiotage Isolera chromatography system. Preparative HPLC purificationswere performed using a Waters preparative HPLC unit equipped with aPhenomenex Kinetex 5 μm EVO C18 150×21.2 mm column. HPLC analyses wereconducted on an Agilent 1100 Series Analytical HPLC equipped with aModel G1322A Degasser, Model G1311A Quarternary Pump, Model G1329AAutosampler, Model G1314 Variable Wavelength Detector, Agilent Poroshell120 SB C18, 4.6 mm×50 mm column at room temperature using a 10-100%gradient of water and acetonitrile containing 0.1% formic acid. HPLCswere monitored at 254 or 205 nm.

Synthesis of HIPS Constructs Bearing Camptothecines

Preparation of (S)-tert-butyl(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (5)

To a solution of SN-38 1 (158 mg, 0.402 mmol) and Boc₂O (114 mg, 1.3mmol) in dichloromethane (16 mL) was added pyridine (0.980 mL, 12.2mmol) at 0° C. After one hour the solution was allowed to warm up toroom temperature and stirred for two hours. The reaction mixture wasthen concentrated under vacuum, and the residue was purified on silicagel (hexane/EtOAc, 100:0 to 0:100 v/v) to yield 5 (160 mg, 80%) as anoff-white solid. LRMS (ESI): m/z 493.2 [M+H]+, Calcd for C₂₇H₂₉N₂O₇ m/z493.2.

Preparation of (S)-tert-butyl(4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-4,9-diyl)(4-nitrophenyl) bis(carbonate) (6)

To a solution of 5 (15 mg, 0.030 mmol) in dichloromethane (1 mL) wasadded nitrophenyl chloroformate (6 mg, 0.030 mmol) and DIPEA (11 mg,0.060 mmol) at 0° C. The solution was allowed to warm to roomtemperature and stirred for 3 hours. The reaction mixture was thenconcentrated and used in the next step without further purification.LRMS (ESI): m/z 658.2 [M+H]⁺, Calcd for C₃₄H₃₂N₃O₁₁ m/z 652.2.

Preparation of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)ethane-1,2-diylbis(methylcarbamate) (8)

To a solution of PNP carbonate 6 (20 mg, 0.030 mmol) and amine 7 (46 mg,0.076 mmol) in DMF (1 mL) was added DIPEA (26 uL, 0.15 mmol). Thereaction mixture was stirred overnight at room temperature and usedfurther in synthesis without purification. LRMS (ESI): m/z 1148.4[M+H]⁺, Calcd for C₆₃H₇₀N₇O₁₄ m/z 1148.5.

Preparation of4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)ethane-1,2-diylbis(methylcarbamate) (9)

To a solution of compound 8 (35 mg, 30 μmol) in DMF (1 mL) was addedpiperidine (100 μL) at room temperature. After 30 minutes, the mixturewas purified by reversed-phase chromatography on C18 column (H₂O/CH₃CNwith 0.05% TFA, 9:1 to 35:65 v/v). The fractions containing the desiredcompound were pooled and concentrated under vacuum to yield compound 9(2.0 mg, 8% yield) as an off-white solid. LRMS (ESI): m/z 826.3 [M+H]⁺,Calcd for C₄₃H₅₂N₇O₁₀ m/z 826.4.

Preparation of(2S,5S,36R)-36-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (11)

To a solution of amine 9 (2 mg, 2.4 μmol) and carboxylic acid 10 (2 mg,2.5 μmol) in DMF (0.5 mL) was added HATU (1.3 mg, 3.3 μmol) and DIPEA(1.3 μL, 7 μmol). The reaction was complete after 30 minutes and thecrude solution of 11 was taken to the next step without furtherpurification. LRMS (ESI): m/z 1603.2 [M+H]⁺, Calcd for C₈₁H₉₆N₁₃O₂₀S m/z1602.7.

Preparation of(2S,5S,36R)-1-((4-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenyl)amino)-36-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (12)

To a crude solution of 11 (˜2.4 μmol) in DMF (0.5 mL) was addedpiperidine (50 μL). After stirring for 15 minutes at room temperature,the reaction mixture was directly purified by reversed phase HPLC usingC18 column (H₂O/CH₃CN with 0.05% TFA, 100:0 to 40:60 v/v). Product 12was obtained as a white solid (0.8 mg, 20% yield). LRMS (ESI): m/z 690.9[M+2H]⁺⁺, Calcd for C₆₆H₈₇N₁₃O₁₈S m/z 690.6.

Preparation of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzoicAcid (15)

To a solution of Fmoc-Val-Ala-OH 13 (38 mg, 93 μmol) and HATU (35 mg, 93μmol) in DMF (1 mL) was added DIPEA (32 uL, 196 μmol). The solution wasstirred for 30 minutes at room temperature, afterwards, 4-amino benzoicacid 14 was added (51 mg, 372 μmol). After stirring for 15 minutes, thereaction mixture was purified by reversed phase chromatography on C18column (H₂O/CH₃CN with 0.05% TFA, 90:10 to 30:70 v/v) to remove excess4-aminobenzoic acid and used in the next step without furtherpurification. LRMS (ESI): m/z 530.2 [M+H]⁺, Calcd for C₃₀H₃₂N₃O₆ m/z530.2.

Preparation of(S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzoate(16)

To the crude carboxylic acid 15 in dichloromethane (2 mL) and DMF (0.5mL) was added Boc-protected SN-38 5 (10 mg, 20 μmol), followed by DCC(38 mg, 180 μmol) and DMAP (16 mg, 130 μmol) at 0° C. After 1 h, thereaction was allowed to warm to room temperature and stirred overnight.The mixture was concentrated under vacuum and used without furtherpurification. LRMS (ESI): m/z 1004.5 [M+H]⁺, Calcd for C₅₇H₅₈N₅O₁₂ m/z1004.4.

Preparation of(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzoate(2S,5S,36R)-36-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonate(R)-1-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-3,6-dioxo-5-(sulfomethyl)-10,13,16,19,22,25,28,31-octaoxa-4,7-diazatetratriacontan-34-oate(17)

To the crude compound 16 were added DMF (1 mL) followed by piperidine(50 μL) at room temperature. The mixture was stirred for 15 minutes andpurified directly by reversed phased HPLC using C18 column (H₂O/CH₃CNwith 0.05% TFA, 100:0 to 30:70 v/v). Fractions containing the desiredcompound were pooled and concentrated under vacuum to yield 17 (3 mg,27% over 2 steps) as an off-white solid. LRMS (ESI): m/z 682.3 [M+H]⁺,Calcd for C₃₇H₄₀N₅O₈ m/z 682.3.

Preparation of(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzoate(2S,5S,36R)-36-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonate(R)-1-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-3,6-dioxo-5-(sulfomethyl)-10,13,16,19,22,25,28,31-octaoxa-4,7-diazatetratriacontan-34-oate(19)

To a solution of amine 17 (3 mg, 4.4 μmol) and carboxylic acid 18 (7 mg,6.6 μmol) in DMF (0.5 mL) was added HATU (2.5 mg, 6.6 μmol) and DIPEA(3.4 μL, 20 μmol). The reaction was complete after 30 minutes, andproduct 19 was used crude without further purification. LRMS (ESI): m/z1722.5 [M+H]⁺, Calcd for C₈₇H₁₀₈N₁₁O₂₄S m/z 1722.7.

Preparation of(2S,5S,36R)-36-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((4-(((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-4-oxobutyl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (20)

To a crude solution of 19 (4.4 μmol) in DMF (0.5 mL) was addedpiperidine (50 μL). After stirring 15 minutes, the solution was purifiedby reversed phase chromatography using C18 column (H₂O/CH₃CN with 0.05%TFA, 100:0 to 40:60 v/v). Product 20 was obtained as a white solid (1.5mg, 21% yield). LRMS (ESI): m/z 1722.5 [M+2H]⁺⁺, Calcd for C₇₂H₉₉N₁₁O₂₂Sm/z 750.8.

Preparation of4-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)butanoicAcid (23)

To a solution of PNP carbonate 21 (100 mg, 0.15 mmol) and4-(methylamino)butyric acid 22 (27 mg, 0.18 mmol) in DCM (1 mL) wereadded DIPEA (72 uL, 0.36 mmol). The reaction was stirred overnight andthen purified by reversed phase chromatography on C18 column (H₂O/CH₃CNwith 0.05% TFA, 100:0 to 0:100 v/v). Product 23 was obtained as a whitesolid (60 mg, 63% yield). LRMS (ESI): m/z 681.3 [M+Na]⁻, Calcd forC₃₆H₄₂N_(4Na)O₈ m/z 681.3.

Preparation of(S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)butanoate(24)

To a solution of carboxylic acid 23 (60 mg, 92 μmol) in dichloromethane(1 mL) and DMF (0.25 mL) was added Boc-protected SN-38 5 (15 mg, 30μmol), followed by DCC (21 mg, 90 μmol), and DMAP (12 mg, 99 μmol) at 0°C. After 1 h, the reaction was allowed to warm up to room temperatureand stirred overnight. Reaction mixture was concentrated under vacuum,and crude product 24 was used without further purification. LRMS (ESI):m/z 1133.4 [M+H]⁺, Calcd for C₆₃H₆₉N₆O₁₄ m/z 1133.5.

Preparation of(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(methyl)amino)butanoate(25)

To the crude compound 24 was added DMF (1 mL) followed by piperidine (50μL). The mixture was stirred for 15 minutes and purified by reversedphased HPLC using C18 column (H₂O/CH₃CN with 0.05% TFA, 100:0 to 40:60v/v). The fractions containing the desired compound were pooled andconcentrated under vacuum to yield 25 (14 mg, 57% over 2 steps) as anoff-white solid. LRMS (ESI): m/z 811.3 [M+H]⁺, Calcd for C₄₃H₅₁N₆O₁₀ m/z811.4.

Preparation of(2S,5S,36R)-36-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (26)

To a mixture of amine 25 (4 mg, 4.9 μmol) and carboxylic acid 18 (7.8mg, 7.4 μmol) in DMF (0.5 mL) were added HATU (2.8 mg, 7.4 μmol) andDIPEA (3.8 μL, 22 μmol) at room temperature. After 30 minutes, reactionmixture was concentrated under vacuum, and crude compound 26 was takento the next step without further purification. LRMS (ESI): m/z 926.6[M+2H]⁺⁺, Calcd for C₉₃H₁₂₀N₁₂O₂₆S m/z 926.9.

Preparation of(2S,5S,36R)-36-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((4-(((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-4-oxobutyl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (27)

To a crude solution of 26 (4.9 μmol) in DMF (0.5 mL) was addedpiperidine (45 μL). After stirring for 15 minutes at room temperature,the reaction mixture was directly purified by reversed phase HPLC on C18column (H₂O/CH₃CN with 0.05% TFA, 100:0 to 40:60 v/v). Product 27 wasobtained as a white solid (3.4 mg, 42% yield). LRMS (ESI): m/z 815.5[M+2H]⁺⁺, Calcd for C₇₈H₁₀₉N₁₂O₂₄S m/z 814.9.

Preparation of(2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-5-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (29)

To a mixture of PNP carbonate 28 (20 mg, 0.020 mmol),N¹,N²-dimethylethane-1,2-diamine (3.3 mg, 0.038 mmol), and HOAT (2.7 mg,0.020 mmol) in DMF (1 mL) were added DIPEA (6.9 uL, 0.040 mmol) at roomtemperature. After stirring the resulting mixture for 2 hours, thesolution was purified by reversed phase HPLC on C18 column (H₂O/CH₃CNwith 0.05% TFA, 9:1 to 25:75 v/v) and semi-pure product 29 was used inthe next step. LRMS (ESI): m/z 962.3 [M+H]⁺, Calcd for C₄₈H₆₀N₅O₁₆ m/z962.4.

Preparation of(2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-5-((((2-(((((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (30)

To a mixture of PNP carbonate 6 (13 mg, 0.020 mmol), crude compound 29,and HOAT (5.4 mg, 0.040 mmol) in DMF (1 mL) were added DIPEA (6.9 uL,0.040 mmol). The reaction was stirred for 1 hour and used withoutfurther purification. LRMS (ESI): m/z 1480.5 [M+H]⁺, Calcd forC₇₆H₈₆N₇O₂₄ m/z 1480.6.

Preparation of(2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-5-((((2-(((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (31)

To a crude solution of 30 in DMF (0.5 mL) was added piperidine (30 μL)at 0° C. After stirring for 2 hours, the reaction mixture was directlypurified by reversed phase HPLC on C18 column (H₂O/CH₃CN with 0.05% TFA,100:0 to 40:60 v/v). Product 31 was obtained as a white solid (5 mg, 22%yield over 3 steps). LRMS (ESI): m/z 1158.4 [M+H]⁺, Calcd forC₅₆H₆₈N₇O₂₀ m/z 1158.5.

Preparationof(2S,5S,18R)-18-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((2-(((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonadecane-19-sulfonicAcid (33)

To a solution of amine 31 (2 mg, 2.6 μmol) and carboxylic acid 10 (2 mg,2.6 μmol) in DMF (0.5 mL) were added HATU (1 mg, 2.6 μmol) and DIPEA(1.3 μL, 7.8 μmol). The reaction was complete after 30 minutes, andcrude compound 33 was used without further purification. LRMS (ESI): m/z1017.1 [M+2H]⁺⁺, Calcd for C₉₄H₁₁₁N₁₃O₃₀S m/z 967.9.

Preparation of(2S,3S,4S,5R,6S)-6-(5-((((2-(((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)-2-((2S,5S,18R)-22-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-isopropyl-2-methyl-4,7,17,20-tetraoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19-tetraazadocosanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (34)

To the crude compound 33 in MeOH (0.6 mL) at 0° C. were added a solutionof LiOH (22 mg) in water (0.9 mL). After stirring reaction mixture for 2h, the mixture was purified by reversed phased HPLC on C18 column(H₂O/CH₃CN with 0.05% TFA, 100:0 to 35:65 v/v). Fractions containing thedesired compound were pooled and concentrated under vacuum to yieldcompound 34 (0.6 mg, 20% over 2 steps) as a white solid. LRMS (ESI): m/z1572.5 [M+H]⁺, Calcd for C₇₂H₉₄N₁₃O₂₅S m/z 1572.6.

Preparation of (9H-fluoren-9-yl)methyl2-((5-amino-1-(3-(tert-butoxy)-3-oxopropyl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazine-1-carboxylate(36)

Nitro compound 35 (116 mg, 0.20 mmol) was dissolved in 1 mL of THF andcombined with a solution of ammonium chloride (85 mg, 1.6 mmol) in 0.5mL of water and 1 mL of methanol. The resulting mixture was vigorouslystirred at room temperature and treated with zinc powder (104 mg, 1.6mmol) in small portions over 5 minutes. Reaction mixture was stirred for2 hours, solids were filtered off, filtrate was diluted with 20 mL ofsaturated aqueous ammonium chloride solution, and extracted with ethylacetate (2×25 mL). Organic extracts were dried over sodium sulfate,solvents removed under vacuum to give crude product 36 which was takento the next step without purification. LRMS (ESI): m/z 555.3 [M+H]⁺,Calcd for C₃₃H₃₈N₄O₄ m/z 555.3.

Preparation of (9H-fluoren-9-yl)methyl2-((1-(3-(tert-butoxy)-3-oxopropyl)-5-(4-(tert-butoxy)-4-oxobutanamido)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazine-1-carboxylate(38)

Crude compound 36 (˜0.20 mmol) was combined with4-(tert-butoxy)-4-oxobutanoic acid 37 (40 mg, 0.23 mmol) in 2 mL of DMF.To this mixture were added DIPEA (0.12 mL, 0.6 mmol), followed by PyAOP(110 mg, 0.21 mmol) in one portion at room temperature. After 30minutes, reaction was quenched by pouring into saturated aqueousammonium chloride, extracted with ethyl acetate, washed with brine,dried over sodium sulfate. Solvent was removed under vacuum to give 120mg (0.17 mmol, 85% yield over 2 steps) of product 38 as a dark oil whichwas used further without additional purification. LRMS (ESI): m/z 733.4[M+Na]⁺, Calcd for C₄₁H₅₀N₄O₇ m/z 733.4.

Preparation of4-((2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1-(2-carboxyethyl)-1H-indol-5-yl)amino)-4-oxobutanoicAcid (39)

Bis-tert-butyl ester compound 38 (120 mg, 0.17 mmol) was dissolved in amixture of 2 mL of anhydrous DCM, 2 mL of TFA, and 0.5 mL oftrisopropylsilane. The resulting mixture was allowed to stand at roomtemperature for 4 hours. Solvents were removed under vacuum, and theresidue was purified by reversed phase chromatography (C18 column, 0-70%v/v gradient of CH₃CN/H₂O with 0.05% TFA) to obtain 53 mg (0.09 mmol,53% yield) of diacid product 39. LRMS (ESI): m/z 599.3 [M+H]⁺, Calcd forC₃₃H₃₄N₄O₇ m/z 599.2.

Preparation of (9H-fluoren-9-yl)methyl1,2-dimethyl-2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-5-(4-oxo-4-(perfluorophenoxy)butanamido)-1H-indol-2-yl)methyl)hydrazine-1-carboxylate(40)

To a mixture of diacid 39 (50 mg, 0.084 mmol) and pentafluorophenol (46mg, 0.25 mmol) in 2 mL of anhydrous THF were added DCC (51 mg, 0.25mmol) in one portion at room temperature. The resulting mixture wasstirred for 16 hours, solids were filtered off, filtrate concentrated,and purified by reversed phased chromatography (C18 column, 0-100% v/vgradient of CH₃CN/H₂O with 0.05% TFA). Fractions containing product wereconcentrated to about 20 mL, poured into 50 mL of 10% aqueous citricacid, and extracted with ethyl acetate (2×20 mL), dried over sodiumsulfate. Solvents were removed under vacuum to give 67 mg (0.072 mmol,86% yield) of bis-pfp ester product 40 as a dark viscous oil. LRMS(ESI): m/z 953.1 [M+Na]⁺, Calcd for C₄₅H₃₂F₁₀N₄O₇ m/z 953.2.

Preparationof(2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-(4-((2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazineyl)methyl)-1-(3-(((S)-1-(((S)-1-((4-((((2-(((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-1H-indol-5-yl)amino)-4-oxobutanamido)-3-methylbutanamido)propanamido)-5-((((2-(((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (41)

To a mixture of bis-PFP ester 40 (1.2 mg, 1.3 μmol), amine 31 (3 mg, 2.6μmol), and HOAT (0.45 mg, 3.3 μmol) in DMF (0.5 mL) were added DIPEA(1.1 uL, 6.5 μmol). The reaction was stirred for 1 hour at roomtemperature. After removing solvent under vacuum, crude intermediate 41was taken to the next step without further purification. LRMS (ESI): m/z1439.9 [M+2H]⁺⁺, Calcd for C₁₄₅H₁₆₆N₁₈O₄₅ m/z 1439.6.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-(4-((1-(3-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-(((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-5-yl)amino)-4-oxobutanamido)-3-methylbutanamido)propanamido)-5-((((2-(((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (42)

To the crude compound 41 in MeOH (0.6 mL) at 0° C. was added a solutionof LiOH (22 mg) in water (0.9 mL). After stirring for 2 h, the mixturewas purified by reversed C18 column chromatography (H₂O/CH₃CN with 0.05%TFA, 100:0 to 35:65 v/v). The fractions containing the desired compoundwere pooled and concentrated under vacuum to yield 42 (0.3 mg, 8% over 2steps) as a white solid. LRMS (ESI): m/z 1188.6 [M+2H]⁺⁺, Calcd forC₁₁₆H₁₄₀N₁₈O₃₇ m/z 1188.5

Preparation of4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate(43)

To a mixture of belotecan hydrochloride (9.4 mg, 20 μmol), HOAT (2.8 mg,20 μmol), and 7 μL of DIPEA (40 μmol) in 1 mL of DMF were added PNPcarbonate 18 (13.6 mg, 20 μmol) in one portion at room temperature.After one hour, 40 μL (0.40 mmol) of piperidine was directly added tothe reaction mixture. After 20 minutes, reaction mixture was purified byreversed phase HPLC (C18 column, 0-50% v/v gradient of CH₃CN/H₂O with0.05% TFA) to give 10.8 mg (14.3 μmol, 72% yield) of compound 43 as ayellow solid. LRMS (ESI): m/z 753.4 [M+H]⁺, Calcd for C₄₁H₄₈N₆O₈ m/z753.4.

Preparation of(2S,5S,36R)-36-(3-(2-((1-(((9H-fluoren-9-yl)methoxy)carbonyl)-2,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (44)

To a mixture of compound 43 (10.8 mg, 14.3 μmol) and carboxylic acid 18(15.2 mg, 14.3 μmol) in 2 mL of DMF were added 8 μL of DIPEA (46 μmol),followed by PyAOP (7.5 mg, 14.3 μmol). After 30 minutes at roomtemperature, reaction mixture was directly purified by reversed phaseHPLC (C18 column, 5-95% v/v gradient of CH₃CN/H₂O with 0.05% TFA).Fractions were lyophilized to give 16.5 mg (9.2 μmol, 64% yield) ofcompound 44 as a yellowish powder. LRMS (ESI): m/z 1794.8 [M+H]⁺, Calcdfor C₉₁H₁₁₆N₁₂O₂₄S m/z 1794.8.

Preparation of(2S,5S,36R)-36-(3-(2-((1,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (45)

To a solution of compound 44 (16.5 mg, 9.2 μmol) in 2 mL of DMA wereadded piperidine (18 μL, 0.18 mmol) at room temperature. After 30minutes, reaction mixture was purified on reversed phase HPLC (C18column, 0-50% v/v gradient of CH₃CN/H₂O with 0.05% TFA). Pure fractionswere lyophilized to give 11.2 mg (7.1 μmol, 77% yield) of compound 45 asa yellow powder. LRMS (ESI): m/z 1572.7 [M+H]⁺, Calcd for C₇₆H₁₀₆N₁₂O₂₂Sm/z 1572.8.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (46)

To a solution of belotecan hydrochloride (20 mg, 43 μmol) in 2 mL DMFwere added 15 uL of DIPEA (86 μmol) and 6 mg of HOAt (43 μmol). Theresulting mixture was combined with PNP carbonate 28 (43 mg, 43 μmol) atroom temperature and stirred for one hour, then DMF was removed undervacuum. The residue was dissolved in 1 mL of MeOH and treated with 1 mLof 1M aqueous LiOH. After 10 minutes, 1 mL of 1M aqueous HCl was addedto the mixture, followed by 1 mL of 0.5M pH 4.7 acetate buffer. Theresulting mixture was stirred for 30 minutes at room temperature anddirectly purified by reversed phase HPLC (C18 column, 0-50% v/v gradientof CH₃CN/H₂O with 0.05% TFA). Solvent was removed under vacuum to give17 mg (18 μmol, 43% yield) of compound 46 as a glassy yellow solid. LRMS(ESI): m/z 945.4 [M+H]⁺, Calcd for C₄₇H₅₆N₆O₁₅ m/z 945.4.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-(4-((1-(3-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-5-yl)amino)-4-oxobutanamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (47)

Compound 46 (17 mg, 18 μmol) was combined with bis-PFP ester 40 (8.3 mg,9 μmol), 2.5 mg of HOAt (18 μmol), and 10 μL of DIPEA (54 μmol) in 2 mLof DMA at room temperature. After one hour, piperidine (35 μL, 0.36mmol) was added to the reaction mixture. After 30 minutes, reactionmixture was directly purified by reversed phase HPLC (C18 column, 0-50%v/v gradient of CH₃CN/H₂O with 0.05% TFA). Lyophilization of purefractions gave 5.0 mg of compound 47 (2.2 μmol, 24% yield) as ayellowish powder. LRMS (ESI): m/z 1116.1 [M+2H]⁺⁺, Calcd forC₁₁₂H₁₃₂N₁₆O₃₃ m/z 1116.0.

Preparation of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(49)

To a stirred mixture of Fmoc-Val-Ala-OH 13 (410 mg, 1.0 mmol) andtert-butyl (4-aminobenzyl)carbamate 48 (267 mg, 1.2 mmol) in 5 mL of DCMand 0.5 mL of MeOH were added EEDQ (495 mg, 2.0 mmol) in one portion atroom temperature. The resulting mixture was stirred in the darkovernight, precipitate was collected by filtration, washed with MTBE,and dried on air to give 520 mg of product 49 (0.85 mmol, 85% yield) asa tan powder. LRMS (ESI): m/z 637.3 [M+Na]⁺, Calcd for C₃₅H₄₂N₄O₆ m/z637.3.

Preparation of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate2,2,2-trifluoroacetate (50)

Boc-protected compound 49 (520 mg, 0.85 mmol) was dissolved in 5 mL ofDCM-TFA mixture (1:1) at room temperature. The reaction mixture wasstirred for 15 minutes, then solvents were removed under vacuum. Theresidue was triturated with 20 mL of MTBE, and the resulting precipitatewas collected by filtration, washed with MTBE, and dried on air to give525 mg of product 50 (0.84 mmol, 99% yield) as a tan powder (TFA salt).LRMS (ESI): m/z 515.3 [M+H]⁺, Calcd for C₃₀H₃₄N₄O₄ m/z 515.3.

Preparation of methyl3-(N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)sulfamoyl)propanoate(5)

To a solution of exatecan mesylate (10 mg, 19 μmole) in 1 mL ofanhydrous DMF were added 10 μL of DIPEA (56 μmole), followed by 4 μL ofsulfonyl chloride 51 (21 μmole) at room temperature. After one hour,reaction mixture was directly purified on reversed phase HPLC (C18column, 0-75% v/v gradient of CH₃CN/H₂O with 0.05% TFA) to give 5 mg (9μmole, 47% yield) of product as a yellow solid. LRMS (ESI): m/z 586.2[M+H]⁺, Calcd for C₂₈H₂₈FN₃O₈S m/z 586.2.

Preparation of3-(N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)sulfamoyl)propanoicAcid (53)

Methyl ester 52 (5 mg, 8.5 μmole) was dissolved in 2 mL of methanol andtreated with 1 mL of 1M aqueous LiOH at room temperature. The resultingmixture was stirred for one hour, quenched with 1 mL of 1M aqueous HCl,followed by 1 mL of 0.5M pH 4.7 acetate buffer. After 10 minutes,reaction mixture was concentrated under vacuum and purified by reversedphase HPLC (C18 column, 0-75% v/v gradient of CH₃CN/H₂O with 0.05% TFA)to give 5.0 mg of carboxylic acid product 53 (8.7 mole, quant.) as abright yellow solid. LRMS (ESI): m/z 572.2 [M+H]⁺, Calcd forC₂₈H₂₈FN₃O₈S m/z 572.6

Preparation of(S)-2-amino-N-((S)-1-((4-((3-(N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)sulfamoyl)propanamido)methyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(54)

To a mixture of carboxylic acid 53 (5.0 mg, 8.7 mole) and amine 50 (5.5mg, 8.7 mole) in 1 mL of DMF were added DIPEA (4.6 μL, 26 mole),followed by PyAOP (4.6 mg, 8.7 mole) in one portion at room temperature.After 30 minutes, reaction mixture was treated with piperidine (17 μL,0.17 mmol), stirred for 15 minutes at room temperature, and purified byreversed phase HPLC (C18 column, 0-50% v/v gradient of CH₃CN/H₂O with0.05% TFA). Solvents were removed under vacuum to obtain 5.0 mg (5.7mole, 66% yield) of compound 54 as a dark yellow oil. LRMS (ESI): m/z846.4 [M+H]⁺, Calcd for C₄₂H₄₈FN₇O₉S m/z 846.3.

Preparation of(2S,5S,36R)-36-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((3-(N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)sulfamoyl)propanamido)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicAcid (55)

To a mixture of compound 54 (5.0 mg, 4.7 mole) and carboxylic acid 18(6.3 mg, 4.7 mole) in 1 mL of DMF were added DIPEA (3 μL, 14 mole),followed by PyAOP (3.1 mg, 4.7 mole) in one portion at room temperature.After 30 minutes, reaction mixture was directly purified by reversedphase HPLC (C18 column, 0-75% v/v gradient of CH₃CN/H₂O with 0.05% TFA)to give 6.0 mg (2.6 mole, 55% yield) of compound 55 as a yellow powder.LRMS (ESI): m/z 1887.8 [M+H]⁺, Calcd for C₉₂H₁₁₆FN₁₃O₂₅S₂ m/z 1886.8.

Preparation of(2S,5S,36R)-36-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-1-((4-((3-(N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)sulfamoyl)propanamido)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontane-37-sulfonicacid (56)

To a solution of compound 55 (6.0 mg, 3.2 mole) in 1 mL of DMA wereadded piperidine (6.3 μL, 63 mole) at room temperature. After 30minutes, reaction mixture was directly purified by reversed phase HPLC(C18 column, 0-50% v/v gradient of CH₃CN/H₂O with 0.05% TFA). Fractionscontaining product were lyophilized to obtain 3.0 mg of compound 56 (1.8mole, 56% yield) as a yellow powder. LRMS (ESI): m/z 1665.7 [M+H]⁺,Calcd for C₇₇H₁₀₆FN₁₃O₂₃S₂ m/z 1664.7.

Preparation of(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (58)

To an oven-dried 20 mL scintillation vial were added Belotecan HCl (2,48 mg, 102 mol) and 1.6 mL of anhydrous DMF, followed by 47 μL of DIPEA(269 μmol) and 13 mg of HOAt (96 μmol). The resulting mixture wastreated with PNP carbonate 57 (104 mg, 101 mmol) as a solid in oneportion at room temperature, stirred overnight. After starting materialwas consumed, 200 μL piperidine (2 mmol) was added. The mixture wasstirred for 30 minutes and was monitored by LC-MS. The reaction mixturewas purified by reversed-phase Biotage® (C18, 0-100% v/v CH₃CN—H₂O with0.05% TFA). Lyophilized pure fractions gave 100 mg of compound 58 (91μmol, 90% yield) as a yellow powder. LRMS (ESI): m/z 1099.4 [M+H]⁺,Calcd for C₅₅H₆₇N₆O₁₈ m/z 1099.5.

Preparation of(2S,5S,18R)-18-amino-1-((4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonadecane-19-sulfonicAcid (60)

To an oven-dried 20 mL scintillation vial were added amine (58, 50 mg,46 μmol), cysteic acid linker (59, 27 mg, 49 μmol), and 0.5 mL ofanhydrous DMF, followed by 24 μL of DIPEA (138 μmol) and 18 mg of HATU(46 μmol). The resulting mixture was stirred at room temperature and wasmonitored by LCMS. After starting material was consumed, the solutionwas concentrated under vacuum to remove DMF. The residue was dissolvedin 1 mL of methanol and slowly treated with 1.5 mL of 1M aqueous LiOHsolution at 0° C. Reaction mixture was stirred for 15 minutes, thenwarmed up to room temperature and stirring continued for 1 hours, untilhydrolysis was judged complete by LCMS analysis. Reaction mixture wasquenched by addition of 1 mL of 1M HCl, followed by 1 mL of 0.5M pH 4.7acetate buffer, concentrated under vacuum, and purified byreversed-phase HPLC (C18, 0-75% v/v CH₃CN—H₂O with 0.05% TFA).Lyophilized pure fractions gave 39 mg of compound 60 (31 μmol, 68%yield) as a yellow powder. LRMS (ESI): m/z 1241.5 [M+H]⁺, Calcd forC₅₇H₇₇N₈O₂₁S m/z 1241.5.

Preparation of(2S,5S,18R)-18-(4-((2-((1,2-dimethylhydrazineyl)methyl)-1-((2S,5S,18R)-1-((4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,17,20-pentaoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19-tetraazadocosan-22-yl)-1H-indol-5-yl)amino)-4-oxobutanamido)-1-((4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonadecane-19-sulfonicAcid (61)

Compound 60 (39 mg, 31 μmol) was combined with bis-PFP ester 40 (14.7mg, 15.5 μmol), 5 mg of HOAt (31 μmol), and 17 μL of DIPEA (93 μmol) in1 mL of DMF at room temperature. After one hour, piperidine 61 μL (0.62mmol) was added to the reaction mixture. After 30 minutes, reactionmixture was directly purified by reversed phase HPLC (C18 column, 0-75%v/v gradient of CH₃CN/H₂O with 0.05% TFA). Lyophilization of purefractions gave 29 mg of compound 61 (10.3 μmol, 66% yield) as ayellowish powder. LRMS (ESI): m/z 1412.1 [M+2H]⁺⁺, Calcd forC₁₃₂H₁₇₄N₂₀O₄₅S₂ m/z 1412.1.

Preparation of perfluorophenyl1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecan-19-oate(63)

In an oven-dried scintillation vial were combined1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecan-19-oicacid (62, 487 mg, 1 mmol) and pentafluorophenol (368 mg, 2 mmol) in 5 mLof anhydrous THF. The resulting mixture was treated with DCC (247 mg,1.2 mmol) in one portion at room temperature, and reaction mixture wasstirred overnight. Precipitated solids were filtered off, solventsremoved under vacuum, and the residue was purified by reversed-phasechromatography (C18 column, 10-100% v/v gradient of CH₃CN/H₂O with 0.05%TFA) to give 670 mg of PFP ester 63 (570 mg, 0.87 mmol, 87% yield) as acolorless oil. LRMS (ESI): m/z 654.2 [M+H]⁺, Calcd for C₃₂H₃₂F₅NO₈ m/z654.2.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazahenicosan-21-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (64)

Compound 46 (262 mg, 0.22 mmol) was dissolved in 4 mL of DMF. To thissolution were added DIPEA (105 μL, 0.66 mmol) and PFP ester 63 (181 mg,0.22 mmol) as a solution in 0.5 mL of DMF, followed by HOAt (38 mg, 0.22mmol). The resulting mixture was allowed to stand at room temperaturefor one hour, then treated directly with 4 mL of triethylamine. Reactionmixture was stirred for 5 hours, until Fmoc-deprotection was complete asjudged by LCMS analysis. Reaction mixture was concentrated under vacuumand purified by reversed-phase chromatography (C18 column, 0-50% v/vgradient of CH₃CN/H₂O with 0.05% TFA) to give 185 mg (0.16 mmol, 73%yield) of compound 64 as a yellow powder. LRMS (ESI): m/z 1192.5 [M+H]⁺,Calcd for C₅₈H₇₇N₇O₂ m/z 1192.5.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S)-25-(5-((2S,5S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazahexacosan-26-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-5-isopropyl-2-methyl-4,7,23-trioxo-10,13,16,19-tetraoxa-3,6,22-triazapentacosanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (65)

Compound 64 (23 mg, 19 μmol) was dissolved in 2 mL of anhydrous DMA. Tothis solution were added DIPEA (10 μL, 57 μmol) and bis-PFP ester 40 (8mg, 8.6 μmol) as solid in one portion at room temperature, followed byHOAt (2.6 mg, 19 μmol). The resulting mixture was allowed to stand atroom temperature for one hour, then treated directly with 17 μL ofpiperidine (172 μmol). After 20 minutes, reaction mixture was purifiedby reversed-phase chromatography HPLC (C18 column, 0-50% v/v gradient ofCH₃CN/H₂O with 0.05% TFA). Pure fractions were lyophilized to give 5.8mg (2.1 μmol, 24% yield) of compound 65 as a yellow powder. LRMS (ESI):m/z 1363.1 [M+2H]⁺⁺, Calcd for C₁₃₄H₁₇₄N₁₈O₄₃ m/z 1362.6.

Preparation of4-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazahenicosan-21-amido)-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate(66)

To a mixture of compounds 58 (30 mg, 27 μmol) and 62 (17 mg, 35 μmol) inDMF (0.5 mL) were added HATU (12 mg, 32 mol), followed by DIPEA (14 μL,82 mol) at room temperature, and the resulting solution was stirred for1 h. Solvent was removed under reduced pressure, and the residue wasdissolved in MeOH (1 mL). To this solution was then added 1 M aqueousLiOH solution (1 mL) at 0° C., and the reaction mixture was allowed toslowly warm up to room temperature. After hydrolysis was judged completeby LCMS analysis, reaction mixture was quenched with pH 4.7 acetatebuffer (1 mL). Solids were filtered off, filtrate was purified byreversed-phase prep HPLC (C18 column, 0-75% acetonitrile-water with0.05% TFA). Pure fractions were collected and lyophilized to giveproduct 66 as a yellow solid (19 mg, 16 μmol, 59% yield). LRMS (ESI):m/z 1178.5 [M+H]⁺, Calcd for C₅₈H₇₉N₇O₁₉ m/z: 1178.5.

Preparation of4-((2S,5S)-25-(2-((1,2-dimethylhydrazinyl)methyl)-5-((2S,5S)-1-((4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazahexacosan-26-amido)-1H-indol-1-yl)-5-isopropyl-2-methyl-4,7,23-trioxo-10,13,16,19-tetraoxa-3,6,22-triazapentacosanamido)-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate(67)

To a solution of compound 66 (19 mg, 16 μmol) in DMF (0.5 mL) were addedDIPEA (9 μL, 48 μmol and HOAt (3 mg, 21 μmol), followed by bis-PFP ester40 (7.4 mg, 8 μmol) at room temperature. The resulting mixture wasstirred for 1 h, until coupling was judged complete by LCMS analysis.Piperidine (32 μL, 0.32 mmol) was then added directly to the reactionmixture at rt, and stirring continued for 15 minutes. Reaction mixturewas then purified by reversed-phase prep HPLC (C18 column, 0-70%acetonitrile-water with 0.05% TFA). Pure fractions were collected andlyophilized to obtain product 67 as a yellow solid (13 mg, 4.8 μmol, 60%yield). LRMS (ESI): m/z 1349.0 [M+2H]²⁺, Calcd for C₁₃₄H₁₇₈N₁₈O₄₁ m/z:1349.1.

Preparation of tert-butyl(S)-14-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-hydroxy-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oate(70)

To a mixture of Fmoc-Glu(OtBu)-OH (68, 42 mg, 0.1 mmol) andamino-PEG4-OH (69, 19 mg, 0.1 mmol) in DMF (1 mL) were added HATU (38mg, 0.1 mmol) and DIPEA (52 μL, 0.3 mmol) at room temperature. Reactionmixture was stirred for 1 h and directly purified by reversed-phasechromatography (C18 column, 0-70% acetonitrile-water with 0.05% TFA).Pure fractions were lyophilized to give compound 70 as a white solid (50mg, 0.83 mmol, 83% yield). LRMS (ESI): m/z 601.3 [M+H]⁺, Calcd forC₃₂H₄₄N₂O₉ m/z: 601.3.

Preparation of(S)-14-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-hydroxy-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oicAcid (71)

Compound 70 (50 mg, 83 μmol) was dissolved in TFA (2 mL) and stirred for1 minute at room temperature. Solvent was removed under reduced pressureand the residue was purified by reversed-phase chromatography (C18column, 0-75% acetonitrile-water with 0.05% TFA). Pure fractions werecollected and lyophilized to obtain compound 71 as a white solid (35 mg,83 μmol, 77% yield). LRMS (ESI): m/z 545.3 [M+H]⁺, Calcd for C₂₈H₃₆N₂O₉m/z: 545.2.

Preparation of4-((14S,19S,22S)-14-amino-1-hydroxy-19-isopropyl-22-methyl-13,17,20-trioxo-3,6,9-trioxa-12,18,21-triazatricosan-23-amido)-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate(72)

To a mixture of amine 58 (30 mg, 27 μmol) and carboxylic acid 71 (15 mg,28 μmol) in DMF (0.5 mL) were added HATU (10 mg, 27 μmol), followed byDIPEA (14 μL, 82 μmol) at room temperature. Reaction mixture was stirredfor 1 h until coupling was found complete by LCMS analysis. Solvent wasremoved under reduced pressure; the residue was dissolved in MeOH (1 mL)and treated with 1M aqueous LiOH solution (1 mL) at 0° C. Reactionmixture was allowed to slowly warm up to room temperature, stirred foradditional 1 h, and quenched with pH 4.7 acetate buffer (1 mL). Solidswere filtered off, and the clear filtrate was purified by reversed-phaseprep HPLC (C18 column, 0-75% acetonitrile-water with 0.05% TFA). Purefractions were combined and lyophilized to give 30 mg (24 μmol, 89%yield) of compound 72 as a yellow solid. LRMS (ESI): m/z 1235.5 [M+H]⁺,Calcd for C₆₀H₈₂N₈O₂ m/z: 1235.6.

Preparation of4-((14S,19S,22S)-14-(4-((2-((1,2-dimethylhydrazinyl)methyl)-1-((S)-14-(3-(((S)-1-(((S)-1-((4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-1-hydroxy-13,16-dioxo-3,6,9-trioxa-12,15-diazaoctadecan-18-yl)-1H-indol-5-yl)amino)-4-oxobutanamido)-1-hydroxy-19-isopropyl-22-methyl-13,17,20-trioxo-3,6,9-trioxa-12,18,21-triazatricosan-23-amido)-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate(73)

To a solution of compound 72 (30 mg, 24 μmol) in DMF (1.0 mL) were addedDIPEA (13 μL, 73 μmol) and HOAt (4.2 mg, 32 μmol), followed by bis-PFPester 40 (11 mg, 12 μmol) in one portion at room temperature. Reactionmixture was allowed to stand for 1 h until reaction was judged completeby LCMS analysis, and treated with piperidine (49 μL, 0.49 mmol) at roomtemperature. Reaction mixture was directly purified by reversed-phaseprep HPLC (C18 column, 0-70% acetonitrile-water with 0.05% TFA). Purefractions were collected and lyophilized to give 24 mg of compound 73 asa yellow solid (8.5 μmol, 70% yield). LRMS (ESI): m/z 1406.3 [M+2H]²⁺,Calcd for C₁₃₈H₁₈₄N₂₀O₄₃ m/z: 1406.2.

Preparationof(2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-5-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (75)

To a round-bottom flask with a stir bar were added alcohol 74 (0.075 g,0.088 mmol) and anhydrous DCM (15 mL), followed by MnO₂ at ambienttemperature in one portion (0.400 g, 4.6 mmol, activated by heatingovernight in an oven @ 130° C.). Reaction mixture was allowed to stirfor 90 minutes, until starting material was completely consumed asjudged by TLC analysis. Reaction mixture was filtered through a pad ofcelite, eluted with DCM. Combined filtrates were concentrated andpurified by silica gel chromatography (0-50% gradient of EtOAc-hexane)to give aldehyde 75 as a white solid (0.057 g, 0.068 mmol, 77% yield).LRMS (ESI): m/z 846.5 [M+H]⁺, Calcd for C₄₃H₄₇N₃O₁₅ m/z: 846.3.

Preparation of(2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-5-(aminomethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (76)

To an oven dried vial with a stir bar were added aldehyde 75 (0.100 g;0.118 mmol) and anhydrous MeOH (10 mL), followed by oven-dried 4Amolecular sieves (˜1 g). The resulting mixture was allowed to stir for10 min at room temperature. Anhydrous ammonium acetate (0.911 g; 11.8mmol) was then added to the mixture and stirring continued for 1 hbefore the addition of sodium cyanoborohydride (0.038 g; 0.591 mmol) inone portion at room temperature. After stirring for additional 1 h,reaction mixture was filtered, concentrated under reduced pressure, andpurified by silica gel chromatography (0-10% MeOH in DCM gradient) togive 0.043 g of amine product 76 (0.051 mmol, 43% yield). LRMS (ESI):m/z 847.4 [M+H]⁺, Calcd for C₄₃H₅₀N₄O₁₄ m/z: 847.3.

Preparation of(S)—N-(2-(4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylglycine(77)

Belotecan-HCl (2, 0.025 g; 0.057 mmol) was dissolved in DMF (0.25 mL)and diluted with MeOH (3.0 mL). The resulting solution was combined withglyoxylic acid (0.011 g; 0.115 mmol) and sodium acetate (0.033 g; 0.40mmol) and stirred for 1 h at room temperature. Reaction mixture was thentreated with sodium cyanoborohydride (0.025 g; 0.40 mmol), stirredovernight at room temperature, and quenched with 1 mL of 0.05% aqueousTFA. Solvents were removed in vacuum to leave crude oil, which waspurified by reversed-phase prep HPLC (C18 column, 5-55%acetonitrile-water/0.05% TFA). Fractions containing the desired productwere collected and lyophilized to give 0.027 g (0.055 mmol, 96% yield)of compound 77 as a pale-yellow solid. LRMS (ESI): m/z 492.2 [M+H]⁺,Calcd for C₂₇H₂₉N₃O₆ m/z: 492.2.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-5-((2-((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)amino)acetamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (78)

To an oven-dried scintillation vial with a stir bar were addedcarboxylic acid 77 (0.018 g; 0.037 mmol) and anhydrous DMF (2 mL),followed by HATU (0.013 g; 0.034 mmol) and DIPEA (30 μL) at roomtemperature. The mixture was allowed to stir for 45 min and thencombined with a mixture amine 76 (0.026 g; 0.030 mmol) and DIPEA (30 μL)in 2 mL of DMF. Reaction mixture was stirred for 1 h, quenched byaddition of aqueous 1% TFA solution (15 mL), transferred to a separatoryfunnel, and extracted with EtOAc. Organic layer was washed with waterand brine, and dried over Na₂SO₄. Removal of solvents under vacuum gavea crude yellow oily solid (0.048 g), which was dissolved in 5 mL of THF.This solution was cooled to 0° C. in an ice bath and slowly treated withchilled aqueous LiOH (1M, 2.0 mL). Reaction mixture was allowed to stirat 0° C. for 1 h, slowly warmed to room temperature, and quenched byadding aqueous HCl (1.0 M) to pH 4. The mixture was purified byreversed-phase prep HPLC (C18 column, 0-50% acetonitrile-water/0.05%TFA) to give 0.020 g of compound 78 (0.021 mmol, 70% yield) as anoff-white solid. LRMS (ESI): m/z 959.1 [M+H]⁺, Calcd for C₄₈H₅₉N₇O₁₄m/z: 958.4

(2S,3S,4S,5R,6S)-6-(2-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazahenicosan-21-amido)-5-((2-((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)amino)acetamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (79)

To a solution of amine 78 (0.020 g, 0.021 mmol) in anhydrous DMF (2 mL)were added PFP ester 63 (0.020 g, 0.031 mmol), HOAt (0.004 g; 0.031mmol), and DIPEA (11 μL) at room temperature. Reaction mixture wasallowed to stir for 45 min, then treated with piperidine (50 μL) andstirred for additional 20 min. The mixture was purified byreversed-phase prep HPLC (C18 column, 0-50% acetonitrile-water/0.05%TFA). Pure fractions were combined and lyophilized to obtain amineproduct 79 as a pale-yellow solid (0.015 g, 0.012 mmol, 57% yield). LRMS(ESI): m/z 1205.5 [M+H]⁺, Calcd for C₅₉H₈₀N₈O₁₉ m/z: 1205.6.

(2S,3S,4S,5R,6S)-6-(2-((2S,5S)-25-(5-((2S,5S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((2-((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)amino)acetamido)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazahexacosan-26-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-5-isopropyl-2-methyl-4,7,23-trioxo-10,13,16,19-tetraoxa-3,6,22-triazapentacosanamido)-5-((2-((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)amino)acetamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (80)

To a solution of amine 79 (15 mg; 12 μmol) in anhydrous DMF (2 mL) wereadded bis-PFP ester 40 (5.5 mg; 6 μmol), followed by HOAt (3.4 mg; 2.5μmol) and DIPEA (22 μL) at room temperature. The resulting mixture wasallowed to stir for 30 min, then 50 μL of piperidine was added, andstirring continued for 20 min. Reaction mixture was diluted with 0.05%TFA (1 mL) and purified by reversed-phase prep HPLC (C18 column, 0-50%acetonitrile-water/0.05% TFA). Pure fractions were collected andimmediately subjected to lyophilization to give 5.2 mg of compound 80 asa yellow solid (1.9 μmol, 32% yield). LRMS (ESI): m/z 1376.2 [M+2H]²⁺,Calcd for C₁₃₆H₁₈₀N₂₀O₄₁ m/z: 1376.1.

Preparation of tert-butyl(S)-22-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-21-oxo-2,5,8,11,14,17-hexaoxa-20-azapentacosan-25-oate(82)

To a round bottom flask with a stir bar were added Fmoc-Glu(OtBu)-OH 68(0.259 g; 0.609 mmol) and DMF (15 mL), followed by HATU (0.215 g; 0.558mmol) and DIPEA (440 μL; 2.54 mmol) at room temperature. The resultingmixture was allowed to stir for 30 min, and combined with mPEG6-amine 81(0.150 g; 0.507 mmol). After 1 h, reaction mixture was transferred to aseparatory funnel, diluted with water (30 mL), and extracted with EtOAc(2×30 mL). Organic layer was washed with water and brine, dried oversodium sulfate. Solvents were removed in vacuum to give 0.50 g of crudeproduct 82 as a colorless oil, which was used further withoutpurification. LRMS (ESI): m/z 703.4 [M+H]⁺, Calcd for C₃₇H₅₄N₂O₁₁ m/z:703.4.

Preparation of(S)-22-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-21-oxo-2,5,8,11,14,17-hexaoxa-20-azapentacosan-25-oicAcid (83)

Crude compound 82 (0.50 g) was dissolved in anhydrous DCM (5 mL) andtreated with TFA (2 mL) at room temperature. Reaction mixture wasallowed to stir for 2 h, then solvents were removed under reducedpressure, and the residue was dried under high vacuum overnight to give0.50 g of crude carboxylic acid 83 as a colorless oil, which was usedfurther without purification. LRMS (ESI): m/z 647.7 [M+H]⁺, Calcd forC₃₃H₄₆N₂O₁₁ m/z: 647.3.

Preparation of perfluorophenyl(S)-22-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-21-oxo-2,5,8,11,14,17-hexaoxa-20-azapentacosan-25-oate(84)

To a stirred solution of crude carboxylic acid 83 (0.50 g) in anhydrousTHF (20 mL) were added pentafluorophenol (1.42 g; 7.73 mmol), followedby DCC (0.32 g; 1.55 mmol) in one portion at room temperature. Reactionmixture was stirred overnight at room temperature, filtered, andconcentrated under vacuum. The residue was then purified by silica gelchromatography (0-10% MeOH in DCM gradient) to give PFP-ester 84 as acolorless solid (0.43 g, 0.53 mmol, 87% yield over 3 steps). LRMS (ESI):m/z 813.7 [M+H]⁺, Calcd for C₃₉H₄₅F₅N₂O₁₁ m/z: 813.3.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((22S,27S,30S)-22-amino-27-isopropyl-30-methyl-21,25,28-trioxo-2,5,8,11,14,17-hexaoxa-20,26,29-triazahentriacontan-31-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (85)

To a solution of compound 46 (30 mg, 31 μmol) in anhydrous DMF (3 mL)were added PFP-ester 84 (31 mg, 38 μmol), followed by HOAt (1.5 mg, 47μmol) and DIPEA (10 μL) at room temperature. Reaction mixture wasstirred for 45 min, then directly treated with piperidine (50 μL). After30 min, reaction mixture was quenched with aqueous 0.05% TFA (1 mL) andpurified by reversed-phase prep HPLC (C18 column, 0-50%acetonitrile-water/0.05% TFA). Fractions containing the desired productwere combined and lyophilized to yield 38 mg of amine 85 as apale-yellow solid (28 μmol, 90% yield). LRMS (ESI): m/z 1351.6 [M+H]⁺,Calcd for C₆₅H₉₀N₈O₂₃ m/z: 1351.6.

(2S,3S,4S,5R,6S)-6-(2-((22S,27S,30S)-22-(3-(5-((S)-22-(3-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-21,24-dioxo-2,5,8,11,14,17-hexaoxa-20,23-diazaheptacosan-27-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)-27-isopropyl-30-methyl-21,25,28-trioxo-2,5,8,11,14,17-hexaoxa-20,26,29-triazahentriacontan-31-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (86)

To a stirred solution of amine 85 (20 mg; 15 μmol) in 3 mL of anhydrousDMF were added bis-PFP ester 40 (6.8 mg; 7.3 μmol), followed by HOAt(2.5 mg; 18 μmol) and DIPEA (13 μL) at room temperature. Reactionmixture was stirred for 30 min and then treated directly with piperidine(50 μL). After 20 min, reaction mixture was purified by reversed-phaseprep HPLC (C18 column, 0-50% acetonitrile-water/0.05% TFA). Purefractions containing product were combined and lyophilized to yield 15mg of compound 86 (5 μmol, 69% yield) as a pale-yellow solid. LRMS(ESI): m/z 1522.2 [M+2H]²⁺, Calcd for C₁₄₈H₂₀₀N₂₀O₄₉ m/z: 1522.2.

Preparation of13-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecan-17-oicAcid (88)

To a stirred solution of compound 87 (100 mg, 186 μmol) in MeCN (2 mL)were added succinic anhydride (93 mg, 928 μmol) and triethylamine (129μL, 928 μmol) at ambient temperature. Reaction mixture was stirred for10 min and then directly purified by reversed-phase chromatography (C18column, 0-50% acetonitrile-water/0.05% TFA). Pure fractions werecollected and lyophilized to obtain compound 88 as a colorless oil (90mg, 141 μmol, 76% yield).

Preparation of (9H-fluoren-9-yl)methyl4-(N-(2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)-4-oxo-4-(perfluorophenoxy)butanamido)piperidine-1-carboxylate(89)

To a mixture of carboxylic acid 88 (90 mg, 141 μmol) andpentafluorophenol (91 mg, 493 μmol) in 2 mL of anhydrous THF were addedDCC (101 mg, 493 μmol) at room temperature. Reaction mixture was stirredovernight, solids were filtered off, solvent was removed under reducedpressure, and the residue was purified by silica gel chromatography(EtOAc-hexane, 0-50% gradient) to yield 42 mg of PFP-ester 89 (52 μmol,37% yield) as an off-white solid.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S)-11-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-5-isopropyl-2,22,22-trimethyl-4,7,10,20-tetraoxo-14,17,21-trioxa-3,6,11-triazatricosanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (90)

To a solution of compound 46 (25 mg, 26 μmol) in DMF (1.0 mL) were addedDIPEA (14 μL, 73 μmol) and HOAt (5 mg, 35 μmol), followed by PFP-ester89 (21 mg, 26 μmol) at room temperature. Reaction mixture was stirredfor 30 min and then directly purified by reversed-phase chromatography(C18, 0-100% v/v MeCN-H₂O with 0.05% TFA). Lyophilized pure fractionsgave 38 mg of compound 90 (24 μmol, 92% yield) as a yellow powder. LRMS(ESI): m/z 1565.7 [M+H]⁺, Calcd for C₈₂H₁₀₀N₈O₂₃ m/z: 1565.7.

Preparation of(2S,5S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,10-tetraoxo-11-(piperidin-4-yl)-14,17-dioxa-3,6,11-triazaicosan-20-oicAcid (91)

A solution of compound 90 (38 mg, 24 μmol) in TFA (2 mL) was stirred forone minute, then diluted with 2 mL of water-acetonitrile mixture (1:1v/v) and lyophilized to give a white solid. The solid was dissolved inDMF (1 mL) and treated with piperidine (49 μL, 0.49 mmol) at roomtemperature. After 20 minutes, reaction mixture was purified byreversed-phase prep HPLC (C18, 0-70% v/v MeCN-H₂O with 0.05% TFA).Lyophilized pure fractions gave 10 mg of compound 91 (7 μmol, 34% yield)as a yellow powder. LRMS (ESI): m/z 1287.5 [M+H]⁺, Calcd for C₆₃H₈₂N₈O₂₁m/z 1287.6.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((15S,18S)-1-carboxy-9-(1-(3-(5-(4-(4-(4-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-N-(2-(2-(2-carboxyethoxy)ethoxy)ethyl)-4-oxobutanamido)piperidin-1-yl)-4-oxobutanamido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-15-isopropyl-18-methyl-10,13,16-trioxo-3,6-dioxa-9,14,17-triazanonadecan-19-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (92)

To a solution of amine 91 (10 mg, 8 μmol) in anhydrous DMF (0.5 mL) wereadded DIPEA (2 μL, 12 μmol) and HOAt (0.7 mg, 5 μmol), followed bycompound 40 (3.5 mg, 4 μmol) in one portion at room temperature.Reaction mixture was stirred for 1 h and then directly treated withpiperidine (8 μL, 160 μmol) at room temperature. After 20 minutes,reaction mixture was purified by reversed-phase prep HPLC (C18, 0-70%v/v MeCN-H₂O with 0.05% TFA). Lyophilized pure fractions gave 2.8 mg ofcompound 92 (1 μmol, 26% yield) as a yellow powder. LRMS (ESI): m/z1458.2 [M+2H]²⁺, Calcd for C₁₄₄H₁₈₄N₂₀O₄₅ m/z 1458.1.

Preparation of (9H-fluoren-9-yl)methyl4-((2,5,8,11-tetraoxatridecan-13-yl)amino)piperidine-1-carboxylate (95)

A mixture of N-Fmoc-piperidone (93, 642 mg, 2 mmol) and mPEG4-amine (94,414 mg, 2 mmol) in DCE (10 mL) was stirred for 30 mins at roomtemperature, and then treated with STAB (840 mg, 4 mmol) in smallportions. The resulting mixture was allowed to stir for 2 h, quenchedwith sat. sodium bicarbonate solution (5 mL), and extracted with EtOAc(3×15 mL). Combined organic layers were washed with brine and dried oversodium sulfate. Solvents were removed in vacuum to give crude product 95as colorless oil (900 mg), which was used further without purification.

Preparation of14-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-15-oxo-2,5,8,11,17-pentaoxa-14-azanonadecan-19-oicAcid (96)

To a solution of crude compound 95 (900 mg) in anhydrous MeCN (10 mL)were added 1,4-dioxane-2,6-dione (1.0 g, 0.93 mmol) and triethylamine(0.85 mL, 0.93 mmol) at room temperature. Reaction mixture was stirredfor 30 min and then directly purified by reversed-phase chromatography(C18, 0-70% v/v MeCN-H₂O with 0.05% TFA). Pure fractions were collectedand lyophilized to obtain compound 96 as a colorless oil (260 mg, 0.45mmol, 23% yield over 2 steps). LRMS (ESI): m/z 629.3 [M+H]⁺, Calcd forC₃₃H₄₄N₂O₁₀ m/z 629.3.

Preparation of (9H-fluoren-9-yl)methyl4-(2-(2-oxo-2-(perfluorophenoxy)ethoxy)-N-(2,5,8,11-tetraoxatridecan-13-yl)acetamido)piperidine-1-carboxylate(97)

To a mixture of acid 96 (260 mg, 0.41 mmol) and pentafluorophenol (264mg, 1.23 mmol) in 2 mL of anhydrous THF were added DCC (253 mg, 1.23mmol) at room temperature. Reaction mixture was stirred overnight,solids were filtered off, solvent was removed under reduced pressure,and the residue was purified by silica gel chromatography (EtOAc-hexane0-50% v/v gradient) to give 163 mg of PFP-ester 97 (0.20 mmol, 50%yield) as a colorless oil. LRMS (ESI): m/z 795.3 [M+H]⁺, Calcd forC₃₉H₄₃F₅N₂O₁₀ m/z 795.3.

Preparation of(2S,3S,4S,5R,6S)-6-(5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-((21S,24S)-21-isopropyl-24-methyl-15,19,22-trioxo-14-(piperidin-4-yl)-2,5,8,11,17-pentaoxa-14,20,23-triazapentacosan-25-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (98)

To a solution of compound 46 (25 mg, 26 μmol) in anhydrous DMF (1.0 mL)were added DIPEA (14 μL, 73 μmol) and HOAt (4.6 mg, 34 μmol) followed byPFP-ester 97 (21 mg, 26 μmol) at room temperature. Reaction mixture wasstirred for 30 min, then piperidine (52 uL, 0.52 mmol) was added, andstirring continued for 20 minutes. Reaction mixture was purifieddirectly by reversed-phase chromatography (C18, 0-100% v/v MeCN-H₂O with0.05% TFA). Lyophilized pure fractions gave 22 mg of compound 98 (16μmol, 62% yield) as a yellow powder. LRMS (ESI): m/z 1333.6 [M+H]⁺,Calcd for C₆₅H₈₈N₈O₂₂ m/z 1333.6.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((21S,24S)-14-(1-(4-((1-(3-(4-(2-(2-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)-N-(2,5,8,11-tetraoxatridecan-13-yl)acetamido)piperidin-1-yl)-3-oxopropyl)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-5-yl)amino)-4-oxobutanoyl)piperidin-4-yl)-21-isopropyl-24-methyl-15,19,22-trioxo-2,5,8,11,17-pentaoxa-14,20,23-triazapentacosan-25-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (99)

To a solution of compound 98 (22 mg, 16 μmol) in anhydrous DMF (1.0 mL)were added DIPEA (4.3 μL, 24 μmol) and HOAt (1.4 mg, 11 μmol), followedby the addition of compound 40 (7 mg, 8 μmol) at room temperature. After30 minutes, piperidine (16 μL, 0.16 mmol) was added in one shot at roomtemperature. Reaction mixture was stirred for 15 minutes and thendirectly purified by reversed-phase prep HPLC (C18, 0-70% v/v MeCN-H₂Owith 0.05% TFA). Lyophilized pure fractions gave 15 mg of 99 (5 μmol,63% yield) as a yellow powder. LRMS (ESI): m/z 1504.2 [M+2H]²⁺, Calcdfor C₁₄₈H₁₉₆N₂₀O₄₇ m/z 1504.2.

Preparation of(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-oxo-3-(perfluorophenoxy)propane-1-sulfonicAcid (101)

To a stirred mixture of Fmoc-L-cysteic acid 100 (100 mg, 0.26 mmol) andpentafluorophenol (94 mg, 0.51 mmol) in 2 mL of anhydrous DMF were addedEDCI-HCl (98 mg, 0.51 mmol) in one portion at room temperature. Theresulting mixture was stirred overnight and then directly purified byreversed-phase chromatography (C18, 0-100% v/v MeCN-H₂O with 0.05% TFA).Pure fractions were concentrated under reduced pressure until solutionbecame murky and lyophilized to give 122 mg of PFP-ester 101 (0.22 mmol,85% yield) as an off-white solid. LRMS (ESI-): m/z 556.2 [M−H]⁻, Calcdfor C₂₄H₁₆F₅NO₇S m/z 556.1.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((R)-2-amino-3-sulfopropanamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (102)

To a mixture of compound 46 (30 mg, 32 μmol) and DIPEA (11 μL, 64 μmol)in 2 mL of anhydrous DMF were added PFP-ester 101 (18 mg, 32 μmol) atroom temperature, followed by HOAt (4.5 mg, 32 μmol). The resultingmixture was allowed to stand at room temperature for 1 h and thentreated with piperidine (63 μL, 0.63 mmol). After 20 minutes, reactionmixture was purified by reversed-phase prep HPLC (C18, 0-50% v/vMeCN-H₂O with 0.05% TFA). Pure fractions containing product werecombined and lyophilized to give 12 mg of compound 102 (11 μmol, 34%yield) as a yellow solid.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((R)-2-(4-((1-(3-(((R)-1-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-1-oxo-3-sulfopropan-2-yl)amino)-3-oxopropyl)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-5-yl)amino)-4-oxobutanamido)-3-sulfopropanamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (103)

To a mixture of compound 102 (12 mg, 11 μmol) and DIPEA (4 μL, 22 μmol)in 2 mL of anhydrous DMF were added bis-PFP-ester 40 (4.5 mg, 5 μmol) atroom temperature, followed by HOAt (1.5 mg, 11 μmol). The resultingmixture was allowed to stand at room temperature for 1 h and thentreated with piperidine (22 μL, 0.22 mmol). After 20 minutes, reactionmixture was purified by reversed-phase prep HPLC (C18, 0-50% v/vMeCN—H₂O/10 mM ammonium formate). Pure fractions containing product werecombined and lyophilized to give 7 mg of compound 103 (2.8 μmol, 56%yield) as a tan powder. LRMS (ESI): m/z 1266.5 [M+2H]²⁺, Calcd forC₁₁₈H₁₄₂N₁₈O₄₁S₂ m/z 1266.5.

Preparation of tert-butyl(S)-16-amino-15-oxo-2,5,8,11-tetraoxa-14-azanonadecan-19-oate (104)

To a solution of Fmoc-Glu-OtBu 68 (0.49 g, 1.2 mmol) in DMF (15 mL) wereadded HATU (0.42 g, 1.1 mmol) and DIPEA (1 mL) at room temperature. Theresulting mixture was stirred for 45 min, then combined with mPEG4-amine94 (0.20 g, 0.96 mmol) and stirred for 30 min at room temperature.Reaction was quenched by addition of 0.05% TFA in water (30 mL) andextracted with EtOAc (2×30 mL). Organic layer was washed with water andbrine, dried over Na₂SO₄, filtered, and concentrated in vacuum to givecrude oil. The crude was resuspended in acetonitrile (20 mL) and treatedwith piperidine (1.0 mL, 1 mmol) at room temperature. After 45 min,solvents were removed in vacuum to give crude oil, which was washed oncewith hexane (10 mL) and purified by reversed-phase chromatography (C18,0-50% v/v MeCN—H₂O with 0.05% TFA). Pure fractions were combined andconcentrated, followed by lyophilization to give amine 104 (0.23 g, 0.57mmol, 58% yield) as an oily solid. LRMS (ESI): m/z 393.3 [M+H]⁺, Calcdfor C₁₈H₃₆N₂O₇ m/z 393.3.

Preparation of tert-butyl(S)-16-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-amido)-15-oxo-2,5,8,11-tetraoxa-14-azanonadecan-19-oate(106)

To a solution of amine 104 (0.23 g; 0.57 mmol) in DMF (10 mL) were addedcarboxylic acid 105 (0.29 g; 0.72 mmol), HATU (0.27 g; 0.69 mmol), andDIPEA (0.50 mL, 2.9 mmol) at room temperature. Reaction mixture wasallowed to stir for 2 h, then poured into 0.05% aqueous TFA (15 mL) andextracted with EtOAc (2×25 mL). Organic layer was washed with water andbrine, and dried over sodium sulfate. Solvents were removed under vacuumto afford crude compound 106 as an oil (0.50 g), which was use furtherwithout purification. LRMS (ESI): m/z 774.9 [M+H]⁺, Calcd forC₄₀H₅₉N₃O₁₂ m/z 774.4.

Preparation of(S)-16-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-amido)-15-oxo-2,5,8,11-tetraoxa-14-azanonadecan-19-oicacid (107)

To a solution of crude ester 106 (0.25 g, 0.32 mmol) in DCM (10 mL) wereadded TFA (4.0 mL), and the resulting solution was allowed to stir atroom temperature for 6h. Solvents were removed in vacuum to give 0.23 g(0.32 mmol, quant. yield) of crude compound 107 as an oil. LRMS (ESI):m/z 718.4 [M+H]⁺, Calcd for C₃₆H₅₁N₃O₁₂ m/z 718.4.

Preparation of perfluorophenyl(S)-16-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-amido)-15-oxo-2,5,8,11-tetraoxa-14-azanonadecan-19-oate(108)

To a solution of crude acid 107 (0.23 g; 0.32 mmol) in anhydrous THF (10mL) were added DCC (0.33 g; 1.57 mmol) and pentafluoro phenol (0.29 g;1.57 mmol) at room temperature. Reaction mixture was allowed to stirovernight at room temperature, then filtered, and concentrated undervacuum. The residue was purified by silica gel chromatography using0-10% MeOH in DCM gradient to give 0.23 g of PFP-ester 106 a colorlessoil (0.23 g, 0.26 mmol, 81% yield). LRMS (ESI): m/z 884.9 [M+H]⁺, Calcdfor C₄₂H₅₀F₅N₃O₁₂ m/z 884.3.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((16R,21S,24S)-16-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-21-isopropyl-24-methyl-15,19,22-trioxo-2,5,8,11-tetraoxa-14,20,23-triazapentacosan-25-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (109)

To a solution of amine 46 (10 mg; 10 μmol) in anhydrous DMF (2 mL) wereadded PFP-ester 106 (11.5 mg; 13 μmol), followed by HOAt (3 mg, 22 μmol)and DIPEA (10 μL) at room temperature. Reaction mixture was allowed tostir for 1 h, then piperidine (50 μL) was added to directly to themixture and stirring continued for 30 mins. Reaction mixture wasquenched by adding 2 mL of aqueous 0.05% TFA solution and purified byreversed-phase prep HPLC (C18, 0-50% v/v MeCN—H₂O with 0.05% TFA). Purefractions were lyophilized to give 13 mg of compound 109 (9 μmol, 90%yield) as a pale-yellow solid. LRMS (ESI): m/z 1422.6 [M+H]⁺, Calcd forC₆₈H₉₅N₉O₂₄ m/z 1422.7.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((16S,21S,24S)-16-(3-(2-(2-(3-(5-((R)-16-(3-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-15,18,28-trioxo-2,5,8,11,21,24-hexaoxa-14,17,27-triazahentriacontan-31-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)ethoxy)ethoxy)propanamido)-21-isopropyl-24-methyl-15,19,22-trioxo-2,5,8,11-tetraoxa-14,20,23-triazapentacosan-25-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (110)

To a solution of amine 110 (13 mg, 9 μmol) in 2.5 mL of anhydrous DMFwere added bis-PFP-ester 40 (4.2 mg, 4.5 μmol), followed by HOAt (2.4mg, 18 μmol) and DIPEA (5 μL). Reaction mixture was allowed to stir for30 mins, then piperidine (50 μL) was added directly to the mixture andstirring continued for 30 min. Reaction mixture was purified byreversed-phase prep HPLC (C18, 0-50% v/v MeCN—H₂O with 0.05% TFA).Lyophilization of pure fractions gave 5 mg of compound 110 (1.6 μmol,36% yield) as a pale-yellow solid. LRMS (ESI): m/z 1593.3 [M+2H]²⁺,Calcd for C₁₅₄H₂₁₀N₂₂O₅₁ m/z 1593.2.

Preparation of(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-oxo-3-((2-(2-(3-oxo-3-(perfluorophenoxy)propoxy)ethoxy)ethyl)amino)propane-1-sulfonicAcid (111)

To a mixture of compound 59 (136 mg, 0.25 mmol) and pentafluorophenol(136 mg, 0.75 mmol) in 2 mL of anhydrous THF were added DCC (155 mg,0.75 mmol) at room temperature. Reaction mixture was stirred overnight,solids were filtered off, solvent was removed under reduced pressure,and the residue was purified by silica gel chromatography (EtOAc-hexane,0-50% v/v gradient) to obtain PFP-ester 111 (27 mg, 38 μmol, 15% yield)as a colorless oil. LRMS (ESI): m/z 717.2 [M+H]⁺, Calcd forC₃₁H₂₉F₅N₂O₁₀S m/z 717.2.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S,18R)-18-amino-5-isopropyl-2-methyl-4,7,17-trioxo-19-sulfo-10,13-dioxa-3,6,16-triazanonadecanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (112)

To a stirred solution of compound 46 (25 mg, 26 μmol) in anhydrous DMF(1.0 mL) were added DIPEA (14 μL, 73 μmol) and HOAt (5 mg, 35 μmol),followed by PFP-ester 111 (19 mg, 27 μmol) in one portion at roomtemperature. Reaction mixture was stirred for 1 h, then purified byreversed-phase chromatography (C18, 0-100% v/v MeCN—H₂O with 0.05% TFA).Lyophilized pure fractions gave 9 mg of compound 112 (7 μmol, 26% yield)as a yellow powder. LRMS (ESI): m/z 1255.4 [M+H]⁺, Calcd forC₅₇H₇₄N₈O₂₂S m/z 1255.5.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S,18R)-23-((1-((2S,5S,18R)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,17,20-pentaoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19-tetraazadocosan-22-yl)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-5-yl)amino)-5-isopropyl-2-methyl-4,7,17,20,23-pentaoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19-tetraazatricosanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (113)

To a solution of compound 112 (9 mg, 7 μmol) in anhydrous DMF (1.0 mL)were added DIPEA (2 μL, 10 μmol) and HOAt (1.4 mg, 10 μmol), followed bybis-PFP ester 40 (3.0 mg, 3.5 μmol) in one portion at room temperature.Reaction mixture was stirred for 30 min, then treated with piperidine (7μL, 70 μmol), let stir for 15 minutes, and then directly purified byreversed-phase prep HPLC (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA).Lyophilized pure fractions gave 6 mg of compound 113 (2.0 μmol, 57%yield) as a yellow powder. LRMS (ESI): m/z 1426.1 [M+2H]²⁺, Calcd forC₁₃₂H₁₆₈N₂₀O₄₇S₂ m/z 1426.0.

Preparation of(S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanamido)ethane-1-sulfonicAcid (114)

To a 100 mL round bottom flask were added Fmoc-Glu(OtBu)-OH 68 (0.750 g,1.77 mmol) and anhydrous DMF (20 mL), followed by HATU (1.02 g, 2.64mmol), HOAt (0.250 g, 2.12 mmol), and DIPEA (500 μL) at roomtemperature. The resulting mixture was stirred for 45 min, then taurine(0.445 g, 3.53 mmol) was added, and the mixture was allowed to stirovernight. Reaction mixture was poured into water and extracted withDCM. Organic layer was washed with water, brine, dried over Na₂SO₄.Solvents were removed in vacuum to give crude compound 114 (1.4 g) as awhite solid. LRMS (ESI−): m/z 531.2 [M−H]⁻, Calcd for C₂₆H₃₂N₂O₈S m/z531.2.

Preparation of(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-oxo-5-((2-sulfoethyl)amino)pentanoicAcid (115)

To a solution of crude compound 114 (1.4 g) in DCM (10 mL) were addedTFA (5 mL) at room temperature. The reaction mixture was allowed to stirovernight, then solvents were removed in vacuum and the residue waspurified by reversed-phase chromatography (C18 column, 0-50% v/vMeCN—H₂O with 0.05% TFA) to give 0.74 g of product 115 as a white solid(1.6 mmol, 88% yield over 2 steps). LRMS (ESI−): m/z 475.1 [M−H]⁻, Calcdfor C₂₂H₂₄N₂O₈S m/z 475.1.

Preparation of(S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-oxo-5-(perfluorophenoxy)pentanamido)ethane-1-sulfonicAcid (116)

To a 100 mL round bottom flask with anhydrous THF (25 mL) were addedcarboxylic acid 115 (0.25 g; 0.53 mmol) and pentafluorophenol (0.49 g;2.6 mmol), followed by DCC (0.83 g; 3.9 mmol) at room temperature. Theresulting mixture was allowed to stir overnight at room temperature,then filtered, concentrated under vacuum, and purified by silica gelchromatography (0-10% MeOH in DCM gradient) to yield 0.18 g of PFP-ester116 as a white solid (0.28 mmol, 53% yield). LRMS (ESI−): m/z 641.1[M−H]⁻, Calcd for C₂₈H₂₃F₅N₂O₈S m/z 641.1.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((R)-4-amino-5-oxo-5-((2-sulfoethyl)amino)pentanamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (117)

To a solution of PFP-ester 116 (30 mg, 48 μmol) in anhydrous DMF (3 mL)were added amine 46 (29 mg, 32 μmol), followed by HOAt (7.4 mg, 54 μmol)and DIPEA (30 μL) at room temperature. The resulting mixture was stirredfor 45 min, then piperidine (50 μL) was added to the mixture andstirring continued for 30 min. Reaction mixture was purified directly byreversed-phase prep HPLC (C18 column, 0-50% v/v MeCN—H₂O with 0.05%TFA). Fractions containing product were concentrated and lyophilized togive 28 mg (24 μmol, 75% yield) of compound 117 as a bright yellowsolid. LRMS (ESI−): m/z 1179.4 [M−H]⁻, Calcd for C₅₄H₆₈N₈O₂₀S m/z1179.4.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((S)-4-(4-((1-(3-(((S)-5-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-1,5-dioxo-1-((2-sulfoethyl)amino)pentan-2-yl)amino)-3-oxopropyl)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-5-yl)amino)-4-oxobutanamido)-5-oxo-5-((2-sulfoethyl)amino)pentanamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (118)

To a solution of compound 117 (28 mg, 24 μmol) in 2 mL of anhydrous DMFwere added bis-PFP-ester 40 (11 mg, 11.6 μmol), followed by HOAt (39 mg,28 μmol) and DIPEA (21 μL). The resulting mixture was stirred for 45min, then treated with piperidine (50 μL), stirred for additional 30min, and purified by reversed-phase prep HPLC (C18 column, 0-50% v/vMeCN—H₂O with 0.05% TFA). Fractions containing the desired product werecollected and lyophilized to give 15 mg of compound 118 as a pale-yellowsolid (5.5 μmol, 47% yield). LRMS (ESI−): m/z 1350.0 [M-2H]²-, Calcd forC₁₂₆H₁₅₆N₂₀O₄₃S₂ m/z 1350.0.

Preparation ofN⁶-(((9H-fluoren-9-yl)methoxy)carbonyl)-N²-(2-sulfoacetyl)-L-lysine(120)

To a solution of 2-sulfoacetic acid (280 mg, 2.0 mmol) in DMF (3 mL)were added HATU (760 mg, 2.0 mmol) and DIPEA (695 μL, 4.0 mmol) at roomtemperature. After stirring this mixture for 30 minutes, amino acid 119(330 mg, 0.90 mmol) was added, and stirring continued for one hour.Reaction mixture was directly purified by reversed phase HPLC using C18column (H₂O/CH₃CN with 0.05% TFA, 90:10 to 0:100 v/v). Fractionscontaining the desired compound were pooled and lyophilized to yieldcompound 1280 mg, 0.57 mmol, 63% yield). LRMS (ESI): m/z 491.2 [M+H]⁺,Calcd for C₂₃H₂₆N₂O₈S m/z 491.1.

Preparation of(S)-2-((6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-oxo-1-(perfluorophenoxy)hexan-2-yl)amino)-2-oxoethane-1-sulfonicAcid (121)

To a stirred mixture of carboxylic acid 120 (280 mg, 0.76 mmol) andpentafluorophenol (315 mg, 1.7 mmol) in DCM (5 mL) was at DCC (35 mg,1.7 mmol) at room temperature. After stirring for one hour, reactionmixture was filtered, concentrated, and purified by reversed phasedchromatography on C18 column (H₂O/CH₃CN with 0.05% TFA, 90:10 to 0:100v/v) to afford compound 121 as a white solid (80 mg, 0.12 mmol, 16%yield). LRMS (ESI): m/z 657.1 [M+H]⁺, Calcd for C₂₉H₂₅F₅N₂O₈S m/z 657.1.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((S)-6-amino-2-(2-sulfoacetamido)hexanamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (122)

To a mixture of amine 46 (10 mg, 11 μmol) and PFP-ester 121 (15 mg, 23μmol) in anhydrous DMF (0.5 mL) were added DIPEA (4.4 μL, 26 μmol) atroom temperature. After stirring overnight, piperidine (50 μL) was addedto the reaction mixture. After stirring for 15 minutes at roomtemperature, reaction mixture was directly purified by reversed phaseprep HPLC using C18 column (H₂O/CH₃CN with 0.05% TFA, 90:10 to 45:55v/v). Fractions containing the desired compound were pooled andlyophilized to yield compound 122 (7 mg, 5.9 μmol, 54% yield). LRMS(ESI): m/z 1195.5 [M+H]⁺, Calcd for C₅₅H₇₀N₈O₂₀S m/z 1195.4.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((S)-6-(4-((1-(3-(((S)-6-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxo-5-(2-sulfoacetamido)hexyl)amino)-3-oxopropyl)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-5-yl)amino)-4-oxobutanamido)-2-(2-sulfoacetamido)hexanamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (123)

To a stirred mixture of amine 122 (7 mg, 6 μmol) and bis-PFP-ester 40(2.6 mg, 2.8 μmol) in DMF (0.5 mL) were added DIPEA (5 μL, 26 μmol) atroom temperature. The resulting mixture was stirred for 2 hours, thenpiperidine (50 μL) was added to the mixture. After stirring for 15minutes at room temperature, the reaction mixture was directly purifiedby reversed phase prep HPLC using C18 column (H₂O/CH₃CN with 0.05% TFA,90:10 to 45:55 v/v). Fractions containing the desired compound werepooled and lyophilized to yield compound 123 (3 mg, 1 μmol, 36% yield).LRMS (ESI): m/z 1365.5 [M+H]²⁺, Calcd for C₁₂₈H₁₆₀N₂₀O₄₃S₂ m/z 1365.5.

Preparation of perfluorophenyl1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31-oate(125)

To a stirred mixture of carboxylic acid 124 (100 mg, 0.15 mmol) andpentafluorophenol (140 mg, 0.75 mmol) in anhydrous THF (2 mL) were addedDCC (37 mg, 0.18 mmol) in one portion at room temperature. The resultingmixture was stirred overnight, filtered, and concentrated under vacuum.The residue was purified by reversed-phase chromatography (C18 column,0-70% v/v MeCN—H₂O with 0.05% TFA) to afford 120 mg of compound 125(0.14 mmol, 93% yield) as a clear colorless oil. LRMS (ESI): m/z 830.3[M+H]⁺, Calcd for C₄₀H₄₈F₅NO₁₂ m/z 830.3.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diazatritriacontan-33-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (126)

A solution of amine 46 (55 mg, 58 μmol) in 2 mL of anhydrous DMF wastreated with DIPEA (20 μL, 0.12 mmol) and HOAt (8 mg, 58 μmol), and thencombined with PFP-ester 125 (48 mg, 58 μmol) in DMF (1 mL) at roomtemperature. The resulting mixture was stirred for 30 minutes, thenpiperidine (115 μL, 115 μmol) was added to the mixture. After 20minutes, reaction mixture was purified by reversed-phase prep HPLC (C18column, 0-50% v/v MeCN—H₂O with 0.05% TFA). Pure fractions containingproduct were combined and lyophilized to give 49 mg of compound 126 as ayellowish solid (36 μmol, 62% yield). LRMS (ESI): m/z 1368.6 [M+H]⁺,Calcd for C₆₆H₉₃N₇O₂₄ m/z 1368.6.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S)-38-((1-((2S,5S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontan-37-yl)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-5-yl)amino)-5-isopropyl-2-methyl-4,7,35,38-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaoctatriacontanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (127)

To a mixture of compound 126 (49 mg, 36 μmol) and DIPEA (13 μL, 72 μmol)in 2 mL of DMA were added bis-PFP-ester 40 (14.6 mg, 16 μmol) in oneportion at room temperature, followed by HOAt (5 mg, 36 μmol). Theresulting mixture was stirred at room temperature for 30 minutes, thenpiperidine (21 μL), was added, and stirring continued for 20 minutes.Reaction mixture was directly purified by reversed-phase prep HPLC (C18column, 0-50% v/v MeCN—H₂O with 0.05% TFA). Lyophilized fractions gave32 mg of compound 127 (10 μmol, 63% yield) as a yellow powder. LRMS(ESI): m/z 1539.3 [M+H]²⁺, Calcd for C₁₅₀H₂₀₆N₁₈O₅ m/z 1538.7.

Preparation ofN⁶-(((9H-fluoren-9-yl)methoxy)carbonyl)-N²-(3-(2-(2-methoxyethoxy)ethoxy)propanoyl)-L-lysine(129)

To a solution of mPEG8-acid 128 (100 mg, 0.24 mmol) in 2 mL of anhydrousDMF were added DIPEA (0.13 mL, 0.72 mmol) and HATU (93 mg, 0.24 mmol) atroom temperature. The resulting mixture was stirred for one hour, thenLys(Fmoc)-OH 119 (89 mg, 0.24 mmol) was added to the mixture, andstirring continued for one hour. Reaction mixture was directly purifiedby reversed-phase chromatography HPLC (C18, 0-70% v/v MeCN—H₂O with0.05% TFA) to give 120 mg of compound 129 (0.16 mmol, 67% yield) as acolorless oil. LRMS (ESI): m/z 763.4 [M+H]⁺, Calcd for C₃₉H₅₈N₂O₁₃ m/z763.4.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-aminobutyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (130)

To a solution of carboxylic acid 129 (45 mg, 59 μmol) in 3 mL ofanhydrous DMF were added DIPEA (21 μL, 120 μmol) and HATU (22 mg, 59μmol) at room temperature. The resulting mixture was stirred for 20minutes and combined with amine 46 (55 mg, 58 μmol) in 1 mL of DMF.Reaction mixture was stirred for 30 minutes, then piperidine (115 μL,1.2 mmol) was added to the mixture at room temperature. After 20minutes, reaction mixture was directly purified by reversed phase prepHPLC (C18, 0-50% v/v MeCN—H₂O with 0.05% TFA). Lyophilization of purefractions afforded 34 mg (23 μmol, 40% yield) of compound 130 as ayellow powder. LRMS (ESI): m/z 1467.7 [M+H]⁺, Calcd for C₇₁H₁₀₂N₈O₂₅ m/z1467.7.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(3-(5-((S)-28-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)-26,34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,33-diazaheptatriacontan-37-amido)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-1-yl)propanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (131)

To a mixture of compound 130 (34 mg, 23 μmol) and DIPEA (8 μL, 46 μmol)in 2 mL of DMA were added bis-PFP ester 40 (9.4 mg, 10.5 μmol), followedby HOAt (3 mg, 23 μmol) at room temperature. The resulting mixture wasallowed to stand for 30 minutes at room temperature, then piperidine (21μL, 0.21 mmol) was added to the mixture at room temperature. After 20minutes, reaction mixture was directly purified by reversed phase prepHPLC (C18, 0-50% v/v MeCN—H₂O with 0.05% TFA). Pure fractions werecombined and lyophilized to afford compound 131 as a yellow solid (23mg, 7 μmol, 67% yield). LRMS (ESI): m/z 1638.3 [M+H]²⁺, Calcd forC₁₆₀H₂₂₄N₂₀O₅₃ m/z 1638.8.

Preparation of(R)-21-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,20-dioxo-3,7,10,13,16-pentaoxa-19-azadocosane-22-sulfonicAcid (133)

To a mixture of Fmoc-L-cysteic acid 100 (391 mg, 1.0 mmol) and amine 132(321 mg, 1.0 mmol) in anhydrous DMF (2 mL) were added HATU (400 mg, 1.05mmol) and DIPEA (0.52 mL, 3 mmol). Reaction mixture was stirred for onehour, and then directly purified by reversed phase chromatography (C18,0-50% v/v MeCN—H₂O with 0.05% TFA) to obtain compound 133 as a colorlessoil (500 mg, 0.72 mmol, 72% yield). LRMS (ESI−): m/z 693.3 [M−H]⁻, Calcdfor C₃₃H₄₆N₂O₁₂S m/z 693.3.

Preparation of(R)-1-(9H-fluoren-9-yl)-3,6-dioxo-5-(sulfomethyl)-2,10,13,16,19-pentaoxa-4,7-diazadocosan-22-oicAcid (134)

To a solution of compound 133 (100 mg, 0.14 mmol) in DCM (2 mL) wereadded TFA (2 mL) at ambient temperature. Reaction mixture was stirredfor 10 minutes, then solvents were removed under vacuum, and the residuewas purified by reversed phase chromatography (C18, 0-75% v/v MeCN—H₂Owith 0.05% TFA) to give compound 134 as a colorless oil (80 mg, 0.12mmol, 86% yield). LRMS (ESI−): m/z 637.2 [M−H]⁻, Calcd for C₂₉H₃₈N₂O₁₂Sm/z 637.2.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S,24R)-24-amino-5-isopropyl-2-methyl-4,7,23-trioxo-25-sulfo-10,13,16,19-tetraoxa-3,6,22-triazapentacosanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (135)

To a solution of compound 134 (9 mg, 14 μmol) in anhydrous DMF (1.0 mL)were added DIPEA (7.4 μL, 42 μmol) and HATU (5 mg, 13 μmol) at roomtemperature. The resulting mixture was stirred for 30 minutes, and thencombined with compound 46 (14 mg, 15 μmol) at room temperature. Afterone hour, piperidine (30 μL) was added to the reaction mixture, andstirring continued for 20 minutes. Reaction mixture was purified byreversed-phase prep HPLC (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA).Lyophilized pure fractions gave 13 mg of compound 135 (10 μmol, 68%yield) as a yellow powder. LRMS (ESI): m/z 1343.5 [M+H]⁺, Calcd forC₆₁H₈₂N₈O₂₄S m/z 1343.5.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S,24R)-28-(5-((2S,5S,24R)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,23,26-pentaoxo-24-(sulfomethyl)-10,13,16,19-tetraoxa-3,6,22,25-tetraazanonacosan-29-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-5-isopropyl-2-methyl-4,7,23,26-tetraoxo-24-(sulfomethyl)-10,13,16,19-tetraoxa-3,6,22,25-tetraazaoctacosanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (136)

To a solution of compound 135 (13 mg, 10 μmol) in anhydrous DMF (0.5 mL)were added DIPEA (5 μL, 15 μmol) and HOAt (2 mg, 15 μmol), followed bybis-PFP ester 40 (4.3 mg, 5 μmol) at room temperature. After 30 minutes,reaction was judged complete by LCMS analysis, and piperidine (10 μL, 97μmol) was added directly to the mixture in one shot at room temperature.After 15 minutes, reaction mixture was purified by reversed phase prepHPLC (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA). Lyophilized purefractions gave 7.4 mg of compound 136 (2.4 μmol, 57% yield) as a yellowpowder. LRMS (ESI): m/z 1514.2.1 [M+2H]²⁺, Calcd for C₁₄₀H₁₈₄N₂₀O₅₁S₂m/z 1514.1.

Preparationof(S)-5-((9H-fluoren-9-yl)methoxy)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-oxopentanoicAcid (138)

To a mixture of Fmoc-Glu-OtBu 137 (426 mg, 1 mmol) and(9H-fluoren-9-yl)methanol (216 mg, 1.1 mmol) in 5 mL of anhydrous THFwere added DCC (247 mg, 1.2 mmol) in one portion at room temperature.The resulting mixture was stirred overnight, filtered, and concentratedunder vacuum. The residue was dissolved in DCM-TFA mixture (1:1 v/v, 6mL) and let stand at room temperature for 30 minutes. Solvents wereremoved under vacuum, the residue was dissolved in 40 mL of EtOAc,washed with sat. ammonium chloride, water, and brine, dried over sodiumsulfate, and purified by silica gel chromatography (0-25% v/vEtOAc-hexane) to afford 230 mg of Fmoc-Glu(OFm)-OH 138 as a colorlesssolid (0.42 mmol, 42% yield). LRMS (ESI): m/z 548.2 [M+H]⁺, Calcd forC₃₄H₂₉NO₆ m/z 548.2.

Preparation of(S)-5-(3-((9H-fluoren-9-yl)methoxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13,16,19-pentaoxa-4,7-diazadocosan-22-oicAcid (139)

To a mixture of Fmoc-Glu(OFm)-OH 138 (230 mg, 0.42 mmol) andamino-PEG4-OtBu 132 (162 mg, 0.46 mmol) in 2 mL of DMF were added DIPEA(0.22 mL, 1.26 mmol), followed by PyAOP (240 mg, 0.42 mmol) at roomtemperature. Reaction mixture was stirred for 30 minutes, then pouredinto sat. ammonium chloride solution and extracted with EtOAc. Organiclayer was washed with brine, and dried over sodium sulfate. Afterremoval of solvents in vacuum, the residue was reconstituted in DCM-TFAmixture (1:1 v/v, 4 mL) at room temperature and stirred for 15 minutes,then solvents were removed in vacuum and the residue was purified byreversed phase chromatography (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA)to give 306 mg of compound 139 as a clear colorless oil (0.39 mmol, 92%yield). LRMS (ESI): m/z 795.3 [M+H]⁺, Calcd for C₄₅H₅₀N₂O₁₁ m/z 795.3.

Preparation of 21-((9H-fluoren-9-yl)methyl)1-(2,3,5,6-tetrafluorophenyl)(S)-18-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-17-oxo-4,7,10,13-tetraoxa-16-azahenicosanedioate(140)

To a mixture of compound 139 (145 mg, 0.18 mmol) and2,3,5,6,-tatrafluorophenol (61 mg, 0.36 mmol) in 2 mL of THF were addedDCC (45 mg, 0.36 mmol) in one portion at room temperature. The resultingmixture was stirred overnight, filtered, concentrated under vacuum andpurified by reversed phase chromatography (C18, 0-80% v/v MeCN—H₂O with0.05% TFA) to give 84 mg of TFP-ester 140 as a colorless oil (0.09 mmol,50% yield). LRMS (ESI): m/z 965.3 [M+Na]⁺, Calcd for C₅₁H₅₀F₄N₂O₁₁ m/z965.3.

Preparation of(2S,5S,24S)-24-amino-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazaheptacosan-27-oicAcid (141)

To a solution of compound 46 (19 mg, 20 μmol) in 3 mL of anhydrous DMFwere added DIPEA (9 μL, 60 μmol) and HOAt (2.7 mg, 20 μmol), followed byTFP ester 140 (19 mg, 20 μmol) in one portion at room temperature.Reaction mixture was stirred for 30 minutes, monitored by LCMS analysis.After reaction was judged complete, piperidine (40 μL) was added to themixture, and stirring continued for 20 minutes. Reaction mixture wasthen purified by reversed-phase prep HPLC (C18, 0-50% v/v MeCN—H₂O with0.05% TFA). Pure fractions were lyophilized to afford 14.6 mg ofcompound 141 as a yellow powder (11 μmol, 55% yield). LRMS (ESI): m/z1321.5 [M+H]⁺, Calcd for C₆₃H₈₄N₈O₂₃ m/z 1321.6.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((2S,5S,24S)-28-(5-((2S,5S,24S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-24-(2-carboxyethyl)-5-isopropyl-2-methyl-1,4,7,23,26-pentaoxo-10,13,16,19-tetraoxa-3,6,22,25-tetraazanonacosan-29-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)-24-(2-carboxyethyl)-5-isopropyl-2-methyl-4,7,23,26-tetraoxo-10,13,16,19-tetraoxa-3,6,22,25-tetraazaoctacosanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (142)

To a solution of compound 141 (14.6 mg, 11 μmol) in 2 mL of DMA wereadded DIPEA (6 μL, 33 μmol) and HOAt (1.5 mg, 11 μmol), followed bybis-PFP ester 40 (4.5 mg, 5 μmol) in one portion at room temperature.The resulting mixture was allowed to stand at room temperature for 30minutes, then piperidine (10 μL) was added directly to the mixture.After 20 minutes, reaction mixture was purified by reversed-phase prepHPLC (C18, 0-50% v/v MeCN—H₂O with 0.05% TFA). Pure fractions werecombined and lyophilized to give 6 mg (2 μmol, 40% yield) of compound142 as a yellow solid. LRMS (ESI): m/z 1491.2 [M+2H]²⁺, Calcd forC₁₄₄H₁₈₈N₂₀O₄₉ m/z 1491.6.

Preparation of(S)-4-ethyl-4,9-dihydroxy-10-((methylamino)methyl)-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(144)

To a solution of 10-hydroxycamtothecin 143 (500 mg, 1.37 mmol) in aceticacid (30 mL) and EtOH (15 mL) were added formaldehyde (1 mL, 37 wt % inH₂O) and MeNH₂ (1 mL, 40% w/w water solution). Reaction mixture wasallowed to stir overnight at room temperature, then concentrated underreduced pressure. The residue was purified by reversed phasechromatography (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA). Pure fractionswere collected and lyophilized to obtain des-Me-topotecan 144 as alight-yellow solid (150 mg, 0.46 mmol, 27% yield). LRMS (ESI): m/z 408.2[M+H]⁺, Calcd for C₂₂H₂₁N₃O₅ m/z 408.2.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-5-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (145)

To a stirred solution of des-Me-topotecan 144 (25 mg, 61 μmol) in DMF(1.5 mL) were added HOAt (8.5 mg, 62 μmol) and DIPEA (30 μL, 184 μmol)at room temperature. The resulting mixture was then treated withPNP-carbonate 28 (58 mg, 62 μmol) in one portion at room temperature.Reaction mixture was stirred overnight until all the starting materialswere consumed as judged by LCMS analysis. Reaction mixture was pouredinto 10 mL of water, and the resulting precipitate was collected anddissolved in THF (2 mL). The THF solution was then treated with aq. LiOH(1 mL, 1M) slowly at 0° C. and stirred for 30 min. Reaction mixture wasallowed to slowly warm to room temperature and stirred for an additionalhour, quenched by adding 1M aq. HCl to pH˜4, filtered, and purified byreversed-phase prep HPLC (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA). Purefractions were collected and lyophilized to obtain compound 145 as ayellow solid (25 mg, 27 μmol, 44% yield). LRMS (ESI): m/z 919.3 [M+H]⁺,Calcd for C₄₄H₅₀N₆O₁₆ m/z 919.3.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-aminobutyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (146)

To a solution of compound 128 (21 mg, 27.5 μmol) in DMF (2 mL) wereadded HATU (10 mg, 31 μmol) and DIPEA (14 μL, 82 μmol) at roomtemperature. The resulting mixture was stirred for one hour, thencompound 145 (25 mg, 27 μmol) was added to the mixture, and stirringcontinued for 1 h, until coupling was judged complete by LCMS analysis.Next, reaction mixture was treated with triethylamine (0.4 mL) andstirred at room temperature for 5 h. Reaction mixture was purified byreversed phase prep HPLC (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA). Purefractions were collected and lyophilized to obtain compound 146 as ayellow solid (26 mg, 18 μmol, 67% yield). LRMS (ESI): m/z 1441.6 [M+H]⁺,Calcd for C₆₈H₉₆N₈O₂₆ m/z 1441.6.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(3-(5-((S)-28-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)-26,34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,33-diazaheptatriacontan-37-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (147)

To a solution of compound 146 (26 mg, 18 μmol) in DMF (1.5 mL) wereadded DIPEA (10 μL, 55 μmol) and HOAt (7 mg, 23 μmol), followed bybis-PFP ester 40 (8.4 mg, 9 μmol) in one portion at room temperature.Reaction mixture was stirred for 30 minutes until coupling was judgedcomplete by LCMS analysis, then diethylamine (37 μL, 0.36 mmol) wasadded to the mixture and stirring continued for 2 hours. Reactionmixture was purified by reversed phase prep HPLC (C18, 0-70% v/vMeCN—H₂O with 0.05% TFA). Pure fractions were collected and lyophilizedto give compound 147 as a yellow solid (18 mg, 6 μmol, 67% yield). LRMS(ESI): m/z 1612.2 [M+2H]²⁺, Calcd for C₁₅₄H₂₁₂N₂₀O₅₅ m/z 1612.2.

Preparation of(S)-4-ethyl-4,9-dihydroxy-10-((isopropylamino)methyl)-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(148)

To a solution of 10-hydroxycamptothecin (500 mg, 1.37 mmol) in HOAc (30mL) and EtOH (15 mL) were added formaldehyde (1 mL, 37 wt % in H₂O) andi-PrNH₂ (150 μL, 1.83 mmol) at room temperature. Reaction mixture wasstirred overnight and then concentrated in vacuum. The residue waspurified by reversed phase chromatography (C18, 0-70% v/v MeCN—H₂O with0.05% TFA). Pure fractions were collected and lyophilized to obtaincompound 148 as an orange solid (200 mg, 0.46 mmol, 36% yield). LRMS(ESI): m/z 436.2 [M+H]⁺, Calcd for C₂₄H₂₅N₃O₅ m/z 436.2.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-5-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (149)

To a solution of compound 148 (50 mg, 115 μmol) in DMF (3 mL) were addedHOAt (16 mg, 115 μmol) and DIPEA (60 μL, 344 μmol) at room temperature.The resulting mixture was treated with PNP-carbonate 28 (116 mg, 115μmol) and stirred at room temperature overnight until all startingmaterials were consumed as judged by HPLC analysis. Reaction mixture wasthen diluted with water (10 mL), the resulting precipitate was collectedand dissolved in THF (3 mL). The THF solution was then treated with aq.LiOH (1 mL, 1M) in at 0° C., stirred for 30 min, warmed up to roomtemperature, and stirred for 1 h. Reaction mixture was purified byreversed phase prep HPLC (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA). Purefractions were combined and lyophilized to give compound 149 as a yellowsolid (31 mg, 33 μmol, 29% yield). LRMS (ESI): m/z 947.4 [M+H]⁺, Calcdfor C₄₆H₅₄N₆O₁₆ m/z 947.4.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-aminobutyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (150)

To a stirred solution of carboxylic acid 129 (31 mg, 41 μmol) inanhydrous DMF (2 mL) were added HATU (15 mg, 36 μmol) and DIPEA (17 μL,94 μmol) at room temperature. The resulting mixture was stirred for 1 h,then compound 149 (31 mg, 33 μmol) was added to the mixture, andstirring continued for 1 h. Next, reaction mixture was directly treatedwith piperidine (62 μL, 0.63 mmol) at room temperature, stirred for 20minutes, and purified by reversed-phase prep HPLC (C18, 0-70% v/vMeCN—H₂O with 0.05% TFA). Pure fractions were collected and lyophilizedto afford compound 150 as a yellow solid (26 mg, 18 μmol, 55% yield).LRMS (ESI): m/z 1469.7 [M+H]⁺, Calcd for C₇₀H₁₀₀N₈O₂₆ m/z 1469.7.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(3-(5-((S)-28-(((S)-1-(((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)-26,34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,33-diazaheptatriacontan-37-amido)-2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((((S)-4-ethyl-4,9-dihydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10-yl)methyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (151)

To a solution of compound 150 (27 mg, 18 μmol) in DMF (1.5 mL) wereadded DIPEA (10 μL, 55 μmol) and HOAt (8 mg, 24 μmol) at roomtemperature, followed by the addition of bis-PFP ester 40 (8 mg, 9 μmol)in one portion. The resulting mixture was stirred for 30 minutes, thenpiperidine (36 μL, 0.36 mmol) was added to the mixture at roomtemperature. After 20 minutes, reaction mixture was purified byreversed-phase prep HPLC (C18, 0-70% v/v MeCN—H₂O with 0.05% TFA). Purefractions were collected and lyophilized to obtain compound 151 as ayellow solid (19 mg, 5.8 μmol, 64% yield). LRMS (ESI): m/z 1640.4[M+2H]²⁺, Calcd for C₁₅₈H₂₂₀N₂₀O₅₅ m/z: 1640.3.

Preparation of(S)-2-amino-N-((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(154)

To a solution of Fmoc-Val-Ala-OH 152 (30 mg, 59 μmol) in DMF (1 mL) wereadded MsCl (13.5 mg, 118 μmol) and DIPEA (20 μL, 118 μmol) at roomtemperature. After one hour, reaction mixture was concentrated andpartitioned between ethyl acetate and saturated sodium bicarbonate.Organic layer was washed with brine and dried over Na₂SO₄. Removal ofsolvents under vacuum gave crude chloride 153, which was dissolved inDMF (1 mL). To this solution were added SN38 (1, 23 mg, 59 μmol) andK₂CO₃ (24 mg, 0.18 mmol), and the reaction mixture was stirredvigorously at 45° C. overnight. Reaction mixture was directly purifiedby reversed phase HPLC using C18 column (H₂O/CH₃CN with 0.05% TFA, 90:10to 45:55 v/v). Fractions containing the desired compound were pooled andlyophilized to yield compound 154 (2.7 mg, 16% yield).

LRMS (ESI): m/z 668.3 [M+H]⁺, Calcd for C₃₇H₄₁N₅O₇ m/z 668.3.

Preparation of(2S,5S,18R)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonadecane-19-sulfonicAcid (155)

To a mixture of amine 154 (8 mg, 9 μmol) and PFP ester 111 (12 mg, 10μmol) in DMF (0.5 mL) were added HOAT (1.2 mg, 9 μmol) and DIPEA (5 μL,27 μmol) at room temperature. The resulting mixture was stirred for 1hour, then DMF (0.5 mL) and piperidine (50 μL) were added to mixture.After stirring for 15 minutes at room temperature, the reaction mixturewas directly purified by reversed phase HPLC using C18 column (H₂O/CH₃CNwith 0.05% TFA, 90:10 to 45:55 v/v). Fractions containing the desiredcompound were pooled and lyophilized to yield compound 155 (3.5 mg, 2.9μmol, 32% yield). LRMS (ESI): m/z 1222.5 [M+H]⁺, Calcd for C₆₀H₇₅N₁₁O₁₅Sm/z 1222.5.

Preparation of(2S,3R,4S,5S,6S)-2-(2-(tert-butoxycarbonyl)-5-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (158)

To a mixture of tert-butyl 2-hydroxy-4-nitrobenzoate 157 (1.57 g, 6.6mmol) and bromide 156 (2.37 g, 6.0 mmol) in 25 mL of acetonitrile wereadded silver(I) oxide (1.53 g, 6.6 mmol). The resulting mixture wasstirred overnight in the dark, then filtered through a pad of silicagel, eluting with ethyl acetate, and concentrated under vacuum. Theresidue was purified by silica gel chromatography (0-10% EtOAc-hexane)to give 2.3 g of compound 158 as a white solid (4.1 mmol, 68% yield).LRMS (ESI): m/z 578.2 [M+Na]⁺, Calcd for C₂₄H₂₉NO₁₄ m/z 578.2.

Preparation of4-nitro-2-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzoicAcid (159)

Compound 158 (180 mg, 0.32 mmol) was dissolved in 4 mL of DCM-TFAmixture (1:1 v/v) at room temperature. The resulting solution wasallowed to stand for 30 minutes, then solvents were removed undervacuum, and the residue was purified by silica gel chromatography (0-5%MeOH-DCM) to give 160 mg of carboxylic acid 159 (0.32 mmol, quant.yield) as a pink foamy solid. LRMS (ESI): m/z 522.1 [M+Na]⁺, Calcd forC₂₄H₂₉NO₁₄ m/z 522.1.

Preparation of(2S,3R,4S,5S,6S)-2-(2-((((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)-5-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (160)

To a solution of carboxylic acid 159 (18 mg, 36 μmol) in dichloromethane(1 mL) and DMF (0.5 mL) were added Boc-protected SN-38 5 (14 mg, 28μmol), followed by DCC (6 mg, 29 μmol) and DMAP (3 mg, 25 μmol) at 0° C.After 1 h, reaction mixture was allowed to warm to room temperature, andstirring continued for 2 h. Reaction mixture was purified byreversed-phase chromatography using C18 column (H₂O/CH₃CN with 0.05%TFA, 100:0 to 0:100 v/v) to yield compound 160 (25 mg, 26 μmol, 93%yield) as a yellow solid. LRMS (ESI): m/z 974.3 [M+H]⁺, Calcd forC₄₇H₄₇N₃O₂₀ m/z 974.3.

Preparation of(2S,3R,4S,5S,6S)-2-(5-amino-2-((((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (161)

To a solution of compound 160 (35 mg, 36 μmol) in EtOAc (0.5 mL) wasadded Pd/C (10 wt %, 2 mg) and triethylamine (2 μL, 22 μmol)). The flaskwas then evacuated and filled with hydrogen gas from a balloon, in threerepeating cycles. Reaction mixture was vigorously stirred for 48 h atroom temperature with H₂ balloon attached. Solids were removed byfiltration through a celite pad, the filtrate was concentrated and driedunder vacuum to give 35 mg of crude compound 161, which was used in thenext step without further purification. LRMS (ESI): m/z 944.3 [M+H]⁺,Calcd for C₄₇H₄₉N₃O₁₈ m/z 944.3.

Preparation of(2S,3R,4S,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (162)

To a mixture of crude amine 161 (35 mg) and Fmoc-Val-Ala-OH 13 (60 mg,0.15 mmol) in DMF (0.5 mL) were added HATU (56 mg, 0.15 mmol) and DIPEA(51 μL, 0.30 mmol) at room temperature. Reaction mixture was stirredovernight and purified by reversed-phase chromatography on C18 column(H₂O/CH₃CN with 0.05% TFA, 100:0 to 0:100 v/v) to yield compound 162 (46mg, 34 μmol, 94% yield over two steps) as a yellow solid. LRMS (ESI):m/z 1336.5 [M+H]⁺, Calcd for C₇₀H₇₃N₅O₂₂ m/z 1336.5.

Preparation of(2S,3S,4S,5R,6S)-6-(5-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (163)

To a solution of compound 162 (20 mg, 15 μmol) in a MeOH-H₂O mixture(4:1 v/v, 1 mL) were added Sc(OTf)₃ (180 mg, 0.36 mmol) at roomtemperature. The resulting mixture was stirred for 2 days andconcentrated under vacuum. The residue was reconstituted inDMF-piperidine mixture (10:1 v/v, 1.1 mL) and stirred for 1 hour at roomtemperature. Reaction mixture was purified by reversed-phasechromatography on C18 column (H₂O/CH₃CN with 0.05% TFA, 90:10 to 35:65v/v) to yield compound 163 (8 mg, 9 μmol, 60% yield). LRMS (ESI): m/z874.3 [M+H]⁺, Calcd for C₄₃H₄₇N₅O₁₅ m/z 874.3.

Preparation of(R)-2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanamido)-3,31-dioxo-31-(perfluorophenoxy)-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontane-1-sulfonicAcid (164)

To a mixture of carboxylic acid 18 (180 mg, 0.17 mmol) andpentafluorophenol (125 mg, 0.68 mmol) in 4 mL of anhydrous THF wereadded DCC (68 mg, 0.33 mmol) at room temperature. The resulting mixturewas stirred overnight, filtered through a pad of celite, concentratedunder vacuum, and purified by reversed phase chromatography (C18, 0-80%acetonitrile-water/0.05% TFA) to give 100 mg of PFP ester 164 as acolorless oil (0.08 mmol, 47% yield). LRMS (ESI): m/z 1225.4 [M+H]⁺,Calcd for C₅₆H₆₉F₅N₆O₁₇S m/z 1225.4.

Preparation of(2S,3S,4S,5R,6S)-6-(2-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)-5-((2S,5S,36R)-40-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-isopropyl-2-methyl-4,7,35,38-tetraoxo-36-(sulfomethyl)-10,13,16,19,22,25,28,31-octaoxa-3,6,34,37-tetraazatetracontanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (165)

To a mixture of amine 163 (8 mg, 9 μmol) and PFP ester 164 (12 mg, 10μmol) in DMF (0.5 mL) were added HOAt (1.2 mg, 9 μmol) and DIPEA (5 μL,27 μmol) at ambient temperature. Reaction mixture was stirred for onehour, then DMF (0.5 mL) was added to the mixture, followed by piperidine(50 μL). After stirring for 15 minutes at room temperature, the reactionmixture was directly purified by reversed phase HPLC using C18 column(H₂O/CH₃CN with 0.05% TFA, 90:10 to 45:55 v/v). Fractions containing thedesired compound were pooled lyophilized to yield compound 165 (3.5 mg,2 μmol, 22% yield) as a yellow solid. LRMS (ESI): m/z 1692.7 [M+H]⁺,Calcd for C₇₈H₁₀₅N₁₁O₂₉S m/z 1692.7.

Preparation of tert-butyl(S)-(2-(4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate(166)

To a mixture of belotecan 2 (50 mg, 0.11 mmol) and Boc₂O (12 mg, 0.23mmol) in dichloromethane (2 mL) were added DIPEA (40 μL, 0.23 mmol) atroom temperature. After stirring for 6 hours, the reaction mixture wasdirectly purified by silica gel chromatography (DCM-MeOH, 100:0 to 95:5v/v) to yield compound 166 (44 mg, 0.08 mmol, 73% yield) as an off-whitesolid. LRMS (ESI): m/z 534.3 [M+H]⁺, Calcd for C₃₀H₃₅N₃O₆ m/z 534.3.

Preparation of(2S,3R,4S,5S,6S)-2-(5-amino-2-((((S)-11-(2-((tert-butoxycarbonyl)(isopropyl)amino)ethyl)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (168)

To a solution of carboxylic acid 159 (240 mg, 480 μmol) indichloromethane (1 mL) and DMF (0.5 mL) were added Boc-protectedbelotecan 166 (100 mg, 190 μmol), followed by DCC (6 mg, 29 μmol) andDMAP (3 mg, 25 μmol) at 0° C. After 1 h, reaction mixture was allowed towarm to room temperature and stirred overnight. The mixture was brieflypurified by passing through a silica gel pad (0-6% MeOH-DCM as aneluent) to give crude compound 167, which was dissolved in EtOAc (2 mL)and combined with Pd/C (10 wt %, 20 mg) and triethylamine (20 μL, 220μmol). Reaction flask was then evacuated and filled with hydrogen gasfrom a balloon, in three repeating cycles. Reaction mixture wasvigorously stirred for 48 h at room temperature with H₂ balloonattached, then filtered through a pad of celite. The filtrate wasconcentrated under vacuum and purified by silica gel chromatography(0-5% MeOH-DCM) to yield compound 168 (60 mg, 61 μmol, 33% yield) as ayellow solid. LRMS (ESI): m/z 985.4 [M+H]⁺, Calcd for C₅₀H₅₆N₄O₁₇ m/z985.4.

Preparation of(2S,3R,4S,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((S)-11-(2-((tert-butoxycarbonyl)(isopropyl)amino)ethyl)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (172)

To a mixture of amine 168 (60 mg, 61 μmol) and Fmoc-Ala-Cl 169 (20 mg,61 μmol) in DMF (1 mL) were added DIPEA (22 μL, 120 μmol) at roomtemperature. Reaction mixture was stirred for 1 h, then DMF (0.5 mL) andpiperidine (50 μL) were added to the mixture. After 30 minutes, thereaction was semi-purified by silica gel chromatography with a gradientof 0 to 5% MeOH in DCM to give crude compound 170. Next, a solution of170 in 1 mL of acetonitrile was treated with Fmoc-Val-OPfp 171 (62 mg,120 μmol) and DIPEA (22 μL, 120 μmol) at room temperature. Afterstirring for 20 minutes, reaction mixture was purified by silica gelchromatography (MeOH-DCM 0-5% gradient) to yield compound 172 (70 mg, 51μmol, 83% yield) as a yellow solid.

LRMS (ESI): m/z 1377.5 [M+H]⁺, Calcd for C₇₃H₈₀N₆O₂₁ m/z 1377.5.

Preparation of(2S,3S,4S,5R,6S)-6-(5-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-((((S)-11-(2-((tert-butoxycarbonyl)(isopropyl)amino)ethyl)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (173)

To a solution of compound 172 (70 mg, 51 μmol) in a MeOH-H₂O mixture(4:1 v/v, 1 mL) were added Sc(OTf)₃ (640 mg, 1.3 mmol) at roomtemperature. Reaction mixture was stirred for two days, thenconcentrated, and reconstituted in DMF-piperidine mixture (10:1 v/v, 1.1mL). Reaction mixture was stirred for 1 hour and purified byreversed-phase chromatography on C18 column (H₂O/CH₃CN with 0.05% TFA,90:10 to 20:80 v/v) to compound 173 (5 mg, 5 μmol, 10% yield). LRMS(ESI): m/z 1015.4 [M+H]⁺, Calcd for C₅₁H₆₂N₆O₁₆ m/z 1015.4.

Preparation of(2S,3S,4S,5R,6S)-6-(5-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diazatritriacontan-33-amido)-2-((((S)-11-(2-((tert-butoxycarbonyl)(isopropyl)amino)ethyl)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (174)

To a mixture of amine 174 (5 mg, 5 μmol) and PFP ester 125 (12 mg, 6μmol) in DMF (0.5 mL) were added DIPEA (5 μL, 29 μmol) at roomtemperature. Reaction mixture was stirred for 1 hour, then DMF (0.5 mL)and piperidine (50 μL) were added to the mixture. After stirring for 15minutes at room temperature, the reaction mixture was directly purifiedby reversed phase prep HPLC using C18 column (H₂O/CH₃CN with 0.05% TFA,90:10 to 30:70 v/v). Fractions containing the desired compound werepooled and lyophilized to yield compound 174 (2 mg, 1.4 μmol, 28%yield). LRMS (ESI): m/z 1438.7 [M+H]⁺, Calcd for C₇₀H₉₉N₇O₂₅ m/z 1438.7.

Preparation of(2S,3S,4S,5R,6S)-6-(5-((2S,5S)-38-((1-((2S,5S)-1-((3-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((S)-4-ethyl-11-(2-(isopropylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontan-37-yl)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-5-yl)amino)-5-isopropyl-2-methyl-4,7,35,38-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaoctatriacontanamido)-2-((((S)-4-ethyl-11-(2-(isopropylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (175)

To a stirred mixture of amine 174 (2 mg, 1.4 μmol) and bis-PFP-ester 40(0.8 mg, 0.7 μmol) in DMF (1 mL) were added DIPEA (0.5 μL, 2.8 μmol) atroom temperature. After 2 hours, reaction mixture was concentrated andthen reconstituted in formic acid (1 mL) at room temperature. After 30minutes, formic acid was removed in vacuum, and the residue wasreconstituted in DMF (1 mL) and piperidine (50 μL). After stirring for15 minutes at room temperature, the reaction mixture was directlypurified by reversed phase HPLC using C18 column (H₂O/CH₃CN with 0.05%TFA, 90:10 to 35:65 v/v). Fractions containing the desired compound werepooled and lyophilized to yield compound 175 (0.7 mg, 0.2 μmol, 33%yield) as a yellow powder. LRMS (ESI): m/z 1509.2 [M+2H]²⁺, Calcd forC₁₄₈H₂₀₂N₁₈O₄₉ m/z 1508.7.

Example 2 Preparation of Conjugates

All synthesized constructs containing camptothecine were conjugated to aset of aldehyde-tagged monoclonal antibodies using HIPS ligation (FIG.2). Analytical characterization of the resulting conjugates is shown inFIGS. 3-69 and FIGS. 106-144.

Bioconjugation, Purification and HPLC Analytics

Aldehyde-tagged antibodies (15 mg/mL) were conjugated to linker-payloads(8 mol. equivalents drug:antibody) for 72 h at 37° C. in 20 mM sodiumcitrate, 50 mM NaCl pH 5.5 containing 0.85% DMA. In some cases, toimprove linker-payload solubility, additional DMA was added up to amaximum of 10% vol/vol. After conjugation, free drug was removed byusing multiple rounds of dilution into 20 mM sodium citrate, 50 mM NaClpH 5.5 and concentration using Amicon 0.5 mL 30 kD MWCO centrifugalfilters (Millipore Sigma #UFC5030BK). To determine the DAR of the finalproduct, ADCs were examined by analytical HIC or PLRP. The HIC column(Tosoh #14947) was run with mobile phase A: 1.5 M ammonium sulfate, 25mM sodium phosphate pH 7.0, and mobile phase B: 25% isopropanol, 18.75mM sodium phosphate pH 7.0. The PLRP column (Agilent #PL1912-1802) wasrun with mobile phase A: 0.1% trifluoroacetic acid in H2O, and mobilephase B: 0.1% trifluoroacetic acid in CH3CN with the column heated to80° C. To determine aggregation, samples were analyzed using analyticalsize exclusion chromatography (SEC; Tosoh #08541) with a mobile phase of300 mM NaCl, 25 mM sodium phosphate pH 6.8, 5% isopropanol.

Results of conjugation of Compounds 47, 61 and 65 to 10 differentantibodies are shown in Table 1 below. Table 1 shows drug-to-antibody(DAR) ratios and % high-molecular weight species (% HMW).

TABLE 1 DAR* % HMW** DAR % HMW DAR % HMW Antibody Compound CompoundCompound Compound Compound Compound Target 47 47 65 65 61 61 Target 16.82 1.8 n.d. n.d. n.d. n.d. Target 2 6.16 6.5 6.78 4.1 6.84 2.3 Target3 4.31 4.3 6.33 6.1 6.88 2.2 Target 4 6.97 0.6 n.d. n.d. n.d. n.d.Target 5 5.45 0.8 6.88 3.3 6.93 3.2 Target 6 7.07 2.0 n.d. n.d. n.d.n.d. Target 7 6.88 1.6 n.d. n.d. n.d. n.d. Target 8 6.1 3.4 6.55 3.57.38 1.4 Target 9 6.84 1.5 6.32 2.2 6.99 3.0 Target 10 5.49 2.7 6.33 6.27.06 2.0 *DAR, drug-to-antibody ratio **HMW, high-molecular weightspecies

Example 3 In Vitro Cytotoxicity Assays

Cell lines were plated in 96-well plates (Costar 3610) at a density of5×104 cells/well in 100 μL of growth media. The next day, cells weretreated with 20 μL of test compounds serially-diluted in media. Afterincubation at 37° C. with 5% CO₂ for 5 days, viability was measuredusing the Promega CellTiter Glo® reagent according to the manufacturer'srecommendations. GI50 curves were calculated in GraphPad Prismnormalized to the payload concentration. Graphs of the cytotoxicityassays (% viability vs. drug concentration (nM)) are shown in FIGS.70-80 and FIGS. 89-105.

Example 4 Rat Pharmacokinetic (PK) Study

Male Sprague-Dawley rats (3 per group) were dosed intravenously with asingle 0.9 mg/kg bolus of test article. K2EDTA-stabilized plasma wascollected at 1 h, 8 h and 24 h, and 2, 4, 6, 8, 10, and 14 dayspost-dose.

PK Sample Analysis

Total antibody and total ADC concentrations were quantified by ELISA asdiagrammed in FIG. 81. For total antibody, conjugates were captured withan anti-human IgG-specific antibody and detected with an HRP-conjugatedanti-human Fc-specific antibody. For total ADC, conjugates were capturedwith an anti-human Fab-specific antibody and detected with a mouseanti-payload primary antibody, followed by an HRP-conjugated anti-mouseIgG-subclass 1-specific secondary antibody. Bound secondary antibody wasdetected using Ultra TMB One-Step ELISA substrate (Thermo Fisher). Afterquenching the reaction with sulfuric acid, signals were read by takingthe absorbance at 450 nm on a Molecular Devices Spectra Max M5 platereader equipped with SoftMax Pro software. Data were analyzed usingGraphPad Prism and Microsoft Excel software.

The results of the PK sample analysis are shown in FIGS. 82-85. FIG. 82shows a graph of concentration (μg/mL) vs. days post dose following a0.9 mg/kg dose of trastuzumab antibody. FIG. 83 shows a graph ofconcentration (μg/mL) vs. days post dose following a 0.9 mg/kg dose of aconventional HER2 topoisomerase inhibitor conjugated ADC bearing aprotease cleavable linker. FIG. 84 shows a graph of concentration(μg/mL) vs. days post dose following a 0.9 mg/kg dose of CH1-3/CT-taggedtrastuzumab conjugated to construct 61. FIG. 85 shows a graph ofconcentration (μg/mL) vs. days post dose following a 0.9 mg/kg dose ofCH1-3/CT-tagged trastuzumab conjugated to construct 65.

Example 5 Xenograft Studies Methods:

NCI-H292 Xenograft: Female SCID Beige mice (7 or 8/group) wereinoculated subcutaneously with 5 million NCI-H292 cells in PBS.Treatment began when the tumors reached an average of 121 mm³ (Day 1).For Study 1, animals were dosed intravenously with vehicle alone,Trodelvy, DS-1062, or with conjugate 3485, a TROP-2 targeted ADCincluding two tag sites conjugated to compound 65 (with a DAR of 6.85).ADCs were dosed at either 10 mg/kg on Days 0, 7, and 21 (Trodelvy) or at6 mg/kg on Days 0 and 21 (DS-1062 and conjugate 3485). For Study 2,animals were dosed intravenously with vehicle alone, DS-1062, or withconjugates 3485, 3789, or 3790, TROP-2 targeted ADCs including two tagsites conjugates to compounds 65, 127, or 131, respectively. The animalswere monitored twice weekly for body weight and tumor size. Animals wereeuthanized when tumors reached 2000 mm³ or body weight loss exceeded15%.

Results:

Results for Study 1 are shown in FIG. 86, which shows a graph of meantumor volume (mm³) vs. days and indicates in vivo efficacy against theNCI-H292 xenograft of TROP-2 targeted ADCs carrying topoisomeraseinhibitor payloads. n=8 mice/group; dosing is indicated by arrows.

Results for Study 2 are shown in FIG. 87, which shows a graph of meantumor volume (mm³) vs. days and indicates in vivo efficacy against anNCI-H292 xenograft of TROP-2 targeted ADCs carrying topoisomeraseinhibitor payloads. n=7 mice/group. A single i.v. dose was delivered onDay 0.

Methods:

NCI-H1781 Xenograft: Female BALB/c nude mice (5/group) were inoculatedsubcutaneously with 20 million NCI-H1781 cells in PBS. Treatment beganwhen the tumors reached an average of 222 mm³ (Day 1). Animals weredosed intravenously with vehicle alone, a nectin-4 Compound 65 conjugatewith a DAR of 6.8, or a nectin-4 mc-GGFG-Dxd conjugate with a DAR of3.7. ADCs were dosed intravenously at 5 mg/kg on Days 0 and 7. Theanimals were monitored twice weekly for body weight and tumor size.Animals were euthanized when tumors reached 2000 mm³ or body weight lossexceeded 15%.

Results:

Results the NCI-H1781 study are shown in FIG. 88, which shows a graph ofmean tumor volume (mm³) vs. days and indicates in vivo efficacy againstan NCI-H1781 xenograft of nectin-4 targeted ADCs carrying topoisomeraseinhibitor payloads. n=5 mice/group. A 5 mg/kg dose was delivered i.v. onDays 0 and 7.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A conjugate of formula (I):

wherein: Z is CR¹⁰ or N, R⁷ is selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl; R⁸ and R⁹ are each independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R⁸ and R⁹ are optionally cyclically linked to form a 5or 6-membered heterocyclyl; each R¹⁰ is independently selected fromhydrogen, halogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl; W is a polypeptide; L is alinker attached to a compound of formula (II) at R¹, R², R³, R⁴, R⁵ orR⁶:

R¹ and R² are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R¹ and R² are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring; R³ and R⁴ are eachindependently selected from hydrogen, halo, hydroxy, amino, substitutedamino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or R³ and R⁴ are optionallycyclically linked to form a 5 or 6-membered cycloalkyl or heterocyclylring; R⁵ is selected from hydrogen, halogen, hydroxy, amino, substitutedamino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl; R⁶ is selected from OH andOC(O)R¹¹; and R¹¹ is selected from hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl,wherein at least one R¹⁰ is optionally linked to a second compound offormula (II).
 2. The conjugate of claim 1, wherein the compound offormula (II) has the structure of formula (IIa):

wherein R³ is OH and L is attached at R⁶; or L is attached at R³ and R⁶is OH; or wherein the compound of formula (II) has the structure offormula (IIb):

wherein R^(1a) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1a) and R⁶ is OH; or wherein the compound offormula (II) has the structure of formula (IIc):

wherein R^(1b) is selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl,carboxyl, carboxyl ester, acyl, and sulfonyl, and L is attached at R⁶;or L is attached at R^(1b) and R⁶ is OH; or wherein the compound offormula (II) has the structure of formula (IId):

wherein R^(2a) and R^(2b) are each independently selected from H, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, carboxyl, carboxyl ester, acyl, and sulfonyl,and L is attached at R⁶; or L is attached at R^(2a) or R^(2b) and R⁶ isOH; or wherein the compound of formula (II) has the structure of formula(IIe):

wherein R^(2c) is selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl, carboxylester, acyl, and sulfonyl, and attachment to L is indicated by the wavyline.
 3. The conjugate of claim 1, wherein L comprises:-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(a)-(T⁵-V⁵)_(e)-(T⁶-V⁶)_(f)-,wherein a, b, c, d, e and f are each independently 0 or 1; T¹, T², T³,T⁴, T⁵ and T⁶ are each independently selected from a covalent bond,(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine (4AP), meta-amino-benzyloxy(MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy(PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB),para-amino-benzylamino (PABA), para-amino-phenyl (PAP),para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide,and an ester, wherein EDA is an ethylene diamine moiety, PEG is apolyethylene glycol, and AA is an amino acid residue or an amino acidanalog, wherein each w is an integer from 1 to 20, each n is an integerfrom 1 to 30, each p is an integer from 1 to 20, and each m is aninteger from 1 to 12; V¹, V², V³, V⁴, V⁵ and V⁶ are each independentlyselected from the group consisting of a covalent bond, —CO—, —NR¹⁵—,—NR¹⁵(CH₂)_(q)—, —NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NRCO—, —C(O)O—, —OC(O)—, —O—,—S—, —S(O)—, —SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and —P(O)OH—, wherein each q isan integer from 1 to 6; each R¹³ is independently selected fromhydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and eachR¹⁵ is independently selected from hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl,carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.
 4. The conjugate of claim 3, wherein: T¹ isselected from a (C₁-C₁₂)alkyl and a substituted (C₁-C₁₂)alkyl; T², T³,T⁴, T⁵ and T⁶ are each independently selected from a covalent bond,(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(m)—, 4-amino-piperidine (4AP), MABO, MABC, PABO,PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester;and V¹, V², V³, V⁴, V⁵ and V⁶ are each independently selected from thegroup consisting of a covalent bond, —CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—,—NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—,—SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂—, and —P(O)OH—; wherein: (PEG)_(n) is

where n is an integer from 1 to 30; EDA is an ethylene diamine moietyhaving the following structure:

where y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine(4AP) is

and each R¹² is independently selected from hydrogen, an alkyl, asubstituted alkyl, a polyethylene glycol moiety, an aryl and asubstituted aryl, wherein any two adjacent R¹² groups may be cyclicallylinked to form a piperazinyl ring.
 5. The conjugate of claim 3, whereinT¹, T², T³, T⁴, T⁵ and T⁶ are each optionally substituted with aglycoside.
 6. The conjugate of claim 3, wherein MABO, MABC, PABO, PABC,PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.7. The conjugate of claim 5, wherein the glycoside is selected from aglucuronide, a galactoside, a glucoside, a mannoside, a fucoside,O—GlcNAc, and O-GalNAc.
 8. The conjugate of claim 3, wherein: T¹ is(C₁-C₁₂)alkyl and V¹ is —CO—; T² is an amino acid analog and V² is —NH—;T³ is (PEG)_(n) and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵ is PABCand V⁵ is absent; and T⁶ is EDA and V⁶ is —CO—; or wherein: T¹ is(C₁-C₁₂)alkyl and V¹ is —CO—; T² is an amino acid analog and V² is —NH—;T³ is (PEG)_(n) and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵ is absentand V⁵ is —NR¹⁵(C₆H₄)—; and T⁶ is absent and V⁶ is —CO—; or wherein: T¹is (C₁-C₁₂)alkyl and V¹ is —CO—; T² is an amino acid analog and V² is—NH—; T³ is (PEG)_(n) and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵ isPABC and V⁵ is —NR¹⁵—; and T⁶ is (C₁-C₁₂)alkyl and V⁶ is —CO—; orwherein: T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—; T² is AA and V² is absent;T³ is PABC and V³ is absent; T⁴ is EDA and V⁴ is —CO—; and e and f areeach 0; or wherein: T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—; T² is an aminoacid analog and V² is —NH—; T³ is (PEG)_(n) and V³ is —CO—; T⁴ is AA andV⁴ is absent; T⁵ is PABC and V⁵ is absent; and f is 0; or wherein: T¹ is(C₁-C₁₂)alkyl and V¹ is —CO—; T² is AA and V² is absent; T³ is PABC andV³ is absent; and d, e and f are each 0; or wherein: T¹ is (C₁-C₁₂)alkyland V¹ is —CO—; T² is an amino acid analog and V² is —NH—; T³ is(PEG)_(n) and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵ is PABA and V⁵is —CO—; and T⁶ is (C₁-C₁₂)alkyl and V⁶ is —SO₂—; or wherein: T¹ is(C₁-C₁₂)alkyl and V¹ is —CONH—; T² is (PEG)_(n) and V² is —CO—; T³ is AAand V³ is absent; T⁴ is PABC and V⁴ is absent; e and f are each 0; orwherein: T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—; T² is substituted(C₁-C₁₂)alkyl and V² is —CO—; T³ is AA and V³ is absent; T⁴ is PABC andV⁴ is absent; e and f are each 0; or wherein: T¹ is (C₁-C₁₂)alkyl and V¹is —CONH—; T² is (PEG)_(n) and V² is —CO—; T³ is AA and V³ is absent; T⁴is PABC and V⁴ is absent; T⁵ is (C₁-C₁₂)alkyl and V⁵ is absent; f is 0;or wherein: T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—; T² is 4AP and V² is—CO—; T³ is (C₁-C₁₂)alkyl and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵is PABC and V⁵ is absent; f is 0; or wherein: T¹ is (C₁-C₁₂)alkyl and V¹is —CO—; T² is 4AP and V² is —CO—; T³ is (C₁-C₁₂)alkyl and V³ is —O—; T⁴is (C₁-C₁₂)alkyl and V⁴ is —CO—; T⁵ is AA and V⁵ is absent; T⁶ is PABCand V⁶ is absent; or wherein: T¹ is (C₁-C₁₂)alkyl and V¹ is —CO—; T² isan amino acid analog and V² is absent; T³ is AA and V³ is absent; T⁴ isPABC and V⁴ is absent; e and f are each 0; or wherein: T¹ is(C₁-C₁₂)alkyl and V¹ is —CONH—; T² is (PEG)_(n) and V² is —CONH—; T³ issubstituted (C₁-C₁₂)alkyl and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵is PABC and V⁵ is absent; f is 0; or wherein: T¹ is (C₁-C₁₂)alkyl and V¹is —CO—; T² is AA and V² is —NH—; T³ is (PEG)_(n) and V³ is —CO—; T⁴ isAA and V⁴ is absent; T⁵ is PABC and V⁵ is absent; f is 0; or wherein: T¹is (C₁-C₁₂)alkyl and V¹ is —CO—; T² is an amino acid analog and V² is—NH—; T³ is (PEG)_(n) and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵ isPABO and V⁵ is absent; and f is 0; or wherein: T¹ is (C₁-C₁₂)alkyl andV¹ is —CO—; T² is an amino acid analog and V² is —NH—; T³ is (PEG)_(n)and V³ is —CO—; T⁴ is AA and V⁴ is absent; T⁵ is PAP and V⁵ is —COO—;and f is 0; or wherein: T¹ is (C₁-C₁₂)alkyl and V¹ is —CONH—; T² is(PEG)_(n) and V² is —CO—; T³ is AA and V³ is absent; T⁴ is PAP and V⁴ is—COO—; and e and f are each
 0. 9. The conjugate of claim 1, wherein oneR¹⁰ is linked via a second linker, L^(B), to a second compound offormula (II).
 10. The conjugate of claim 9, wherein L^(B) comprises:-(T⁷-V⁷)_(g)-(T⁸-V⁸)_(h)-(T⁹-V⁹)_(i)-(T⁰°V¹⁰)_(j)-(T¹¹-V¹¹)_(k)-(T¹²-V¹²)_(l)-, wherein g, h, i, j, k and l areeach independently 0 or 1; T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² are eachindependently selected from a covalent bond, (C₁-C₁₂)alkyl, substituted(C₁-C₁₂)alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, (EDA)_(w), (PEG)_(n), (AA)_(p),—(CR¹³OH)_(m)—, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO),meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO),para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB),para-amino-benzylamino (PABA), para-amino-phenyl (PAP),para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide,and an ester, wherein EDA is an ethylene diamine moiety, PEG is apolyethylene glycol, and AA is an amino acid residue or an amino acidanalog, wherein each w is an integer from 1 to 20, each n is an integerfrom 1 to 30, each p is an integer from 1 to 20, and each m is aninteger from 1 to 12; V⁷, V⁸, V⁹, V¹⁰, V¹¹ and V¹² are eachindependently selected from the group consisting of a covalent bond,—CO—, —NR¹⁵—, —NR¹⁵(CH₂)_(q)—, —NR¹⁵(C₆H₄)—, —CONR¹⁵—, —NR¹⁵CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹⁵—, —NR¹⁵SO₂— and—P(O)OH—, wherein each q is an integer from 1 to 6; each R¹³ isindependently selected from hydrogen, alkyl, substituted alkyl, aryl,and substituted aryl; and each R¹⁵ is independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 11.The conjugate of claim 10, wherein T⁷, T⁸, T⁹, T¹⁰, T¹¹ and T¹² are eachoptionally substituted with a glycoside.
 12. The conjugate of claim 10,wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are eachoptionally substituted with a glycoside.
 13. The conjugate of claim 11,wherein the glycoside is selected from a glucuronide, a galactoside, aglucoside, a mannoside, a fucoside, 0-GlcNAc, and O-GalNAc.
 14. Theconjugate of claim 10, wherein: T⁷ is absent and V⁷ is —NHCO—; T⁸ is(C₁-C₁₂)alkyl and V⁸ is —CO—; T⁹ is AA and V⁹ is absent; T¹⁰ is PABC andV¹⁰ is absent; T¹¹ is EDA and V¹¹ is —CO—; and l is 0; or wherein: T⁷ isabsent and V⁷ is —NHCO—; T⁸ is (C₁-C₁₂)alkyl and V⁸ is —CO—; T⁹ is AAand V⁹ is absent; T¹⁰ is PABC and V¹⁰ is absent; and k and l are each 0;or wherein: T⁷ is absent and V⁷ is —NHCO—; T⁸ is (C₁-C₁₂)alkyl and V⁸ is—CO—; T⁹ is an amino acid analog and V⁹ is —NH—; T¹⁰ is (PEG)_(n) andV¹⁰ is —CO—; T¹¹ is AA and V¹¹ is absent; and T¹² is PABC and V¹² isabsent; or wherein: T⁷ is absent and V⁷ is —NHCO—; T⁸ is (C₁-C₁₂)alkyland V⁸ is —CONH—; T⁹ is (PEG)_(n) and V⁹ is —CO—; T¹⁰ is AA and V¹⁰ isabsent; T¹¹ is PABC and V¹¹ is absent; and l is 0; or wherein: T⁷ is(C₁-C₁₂)alkyl and V⁷ is —CONH—; T⁸ is substituted (C₁-C₁₂)alkyl and V⁸is —CO—; T⁹ is AA and V⁹ is absent; T¹⁰ is PABC and V¹⁰ is absent; k andl are each 0; or wherein: T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—; T⁸ is(PEG)_(n) and V⁸ is —CO—; T⁹ is AA and V⁹ is absent; T¹⁰ is PABC and V¹⁰is absent; T¹¹ is (C₁-C₁₂)alkyl and V¹¹ is absent; l is 0; or wherein:T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—; T⁸ is 4AP and V⁸ is —CO—; T⁹ is(C₁-C₁₂)alkyl and V⁹ is —CO—; T¹⁰ is AA and V¹⁰ is absent; T¹¹ is PABCand V¹¹ is absent; l is 0; or wherein: T⁷ is (C₁-C₁₂)alkyl and V⁷ is—CO—; T⁸ is 4AP and V⁸ is —CO—; T⁹ is (C₁-C₁₂)alkyl and V⁹ is —O—; T¹⁰is (C₁-C₁2)alkyl and V¹⁰ is —CO—; T¹¹ is AA and V¹¹ is absent; T¹² isPABC and V¹² is absent; or wherein: T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CO—;T⁸ is an amino acid analog and V⁸ is absent; T⁹ is AA and V⁹ is absent;T¹⁰ is PABC and V¹⁰ is absent; k and l are each 0; or wherein: T⁷ is(C₁-C₁₂)alkyl and V⁷ is —CONH—; T⁸ is (PEG)_(n) and V⁸ is —CONH—; T⁹ issubstituted (C₁-C₁₂)alkyl and V⁹ is —CO—; T¹⁰ is AA and V¹⁰ is absent;T¹¹ is PABC and V¹¹ is absent; l is 0; or wherein: T⁷ is (C₁-C₁₂)alkyland V⁷ is —CO—; T⁸ is AA and V⁸ is —NH—; T⁹ is (PEG)_(n) and V⁹ is —CO—;T¹⁰ is AA and V¹⁰ is absent; T¹¹ is PABC and V¹¹ is absent; l is 0; orwherein: T⁷ is (C₁-C₁₂)alkyl and V⁷ is —CONH—; T⁸ is (PEG)_(n) and V⁸ is—CO—; T⁹ is AA and V⁹ is absent; T¹⁰ is PAP and V¹⁰ is —COO—; and k andl are each
 0. 15. The conjugate of claim 1, wherein the conjugate isselected from:


16. A compound of formula (III):

wherein: Z is CR¹⁰ or N, R⁸ and R⁹ are each independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, or R⁸ and R⁹ are optionally cyclically linkedto form a 5 or 6-membered heterocyclyl; each R¹⁰ is independentlyselected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl; L is a linker attached to acompound of formula (II) at R¹, R², R³, R⁴, R⁵ or R⁶:

R¹ and R² are each independently selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl, or R¹ and R² are optionally cyclically linked to form a 5or 6-membered cycloalkyl or heterocyclyl ring; R³ and R⁴ are eachindependently selected from hydrogen, halogen, hydroxy, amino,substituted amino, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR³ and R⁴ are optionally cyclically linked to form a 5 or 6-memberedcycloalkyl or heterocyclyl ring; R⁵ is selected from hydrogen, halogen,hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl; R⁶ is selected from OH and OC(O)R¹¹; and R¹¹ is selectedfrom hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, wherein at least one R¹⁰ isoptionally linked to a second compound of formula (II). 17.-30.(canceled)
 31. A pharmaceutical composition comprising: a conjugate ofclaim 1; and a pharmaceutically-acceptable excipient.
 32. A methodcomprising: administering to a subject an effective amount of aconjugate of claim
 1. 33. A method of treating cancer in a subject, themethod comprising: administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a conjugateof claim 1, wherein the administering is effective to treat cancer inthe subject.